Shortly after beginning his training Vladimir Komarov was hospitalised for a minor operation in May 1960, which left him medically unfit for physical training for approximately 6 months. At the time, the selection criteria placed a heavy emphasis on the physical condition of cosmonauts and any imperfection led to instant disqualification. Since Komarov already held engineering qualifications, he was allowed to remain a part of the program after assuring the administration he would be able to catch up. He continued with the required academic studies while recovering. He returned to training in October, because his recovery was more rapid than medical staff had expected. During that time he assisted his younger peers with their academic studies; earning him the casual nickname of “The Professor” which he shared with Belyayev who was two years his senior. In 1961 the first space flights began. By 1962, Komarov was the third highest paid cosmonaut, due to his qualifications, rank and experience. He earned 528 rubles a month, with only cosmonauts 1 and 2 Yuri Gagarin and Gherman Titov being more highly paid. When Georgi Shonin demonstrated an unacceptable level of g-force susceptibility in the centrifuge he was replaced by Komarov in May 1962 for planned dual Vostok missions. Komarov was selected as back up for Pavel Popovich (Vostok 4), but subsequent routine ECG testing of Komarov revealed a heart irregularity and he was pulled from the program and replaced by Boris Volynov. The same heart irregularity grounded American astronaut Deke Slayton. After Komarov persistently lobbied medical and military personnel to be readmitted into the program he was allowed to return to training.

In 1963, cosmonaut training was conducted in 6 Groups, with Vladimir Komarov being selected in Group 2 with Valery Bykovsky and Volynov. This group was to train for missions of up to five days in duration scheduled for the latter part of 1963. In May 1963 Alekseyev proposed to Kamanin that Komarov be named backup for Vostok 5 rather than Khrunov because his suit was ready. Komarov was later named in a further group for planned missions in 1964 with Belyaev, Shonin, Khrunov, Zaikin, Gorbatko, Volynov, and Leonov. The training groups were formed for later Vostok missions, (Vostok 7–13) but no actual crews were assigned and the missions did not occur under the auspices of the original Vostok program. In December 1963, Komarov was shortlisted for flight by Kamanin with Volynov and Leonov, having completed two years of training. In April 1964 Vladimir Komarov was declared space-flight ready with Bykovsky, Popovich, Titov, Volynov, Leonov, Khrunov, Belyayev, and Lev Demin. From this group the commander of the planned Voskhod mission scheduled for late 1964 would be chosen. In May the group was reduced to Volynov, Komarov, Leonov, Khrunov. During training, Vladimir Komarov lived at the TsPK (which would later be nicknamed Star City by the Soviet press) with his wife Valentina and their two children Yevgeny and Irina. There he enjoyed hunting, cross country skiing, ice hockey and other social activities with his fellow trainees in their leisure time. Komarov was well liked by his peers and was referred to by them as Volodya (a diminutive of his first name). Pavel Popovich noted that Komarov was respected for his humility and experience: “…he was already an engineer when he joined us, but he never looked down on the others. He was warm-hearted, purposeful and industrious. Volodya’s prestige was so high that people came to him to discuss all questions: personal as well as questions of our work.” Fellow cosmonaut Alexei Leonov described him as “…very serious. He was a first-class test pilot.”

Vladimir Komarov was assigned to the Soviet Soyuz program along with Yuri Gagarin and Alexei Leonov. In July 1966, Komarov was reprimanded by Kamanin for his unauthorised disclosure, while in Japan, that “the Soviet Union will, at the scheduled time, fly an automated spacecraft around the Moon and return it to (the) Earth, to be followed by a dog flight, then a manned circumlunar flight.” The following month Komarov clashed with other engineers over ongoing design problems in which zero-G tests showed that the Soyuz module hatch was too small to allow the exit of a fully suited cosmonaut safely. Meanwhile Komarov and his fellow cosmonauts had their groups and assignments constantly revised and they became increasingly anxious about the lack of response to their concerns about the design and manufacture of space craft which had been raised in a letter to Leonid Brezhnev by Yuri Gagarin on their behalf. Komarov was selected to command the Soyuz 1, in 1967, with Yuri Gagarin as his backup cosmonaut. During the preparations for the spaceflight, both cosmonauts were working twelve- to fourteen-hour days. On orbital insertion, the solar panels of the Soyuz module failed to fully deploy thereby preventing the craft from being fully powered and obscuring some of the navigation equipment. Komarov reported: “Conditions are poor. The cabin parameters are normal, but the left solar panel didn’t deploy. The electrical bus is at only 13 to 14 amperes. The HF (high frequency) communications are not working. I cannot orient the spacecraft to the sun. I tried orienting the spacecraft manually using the DO-1 orientation engines, but the pressure remaining on the DO-1 has gone down to 180.” Komarov tried unsuccessfully to orient the Soyuz module for five hours. The craft was transmitting unreliable status information and communications were lost on orbits 13 through 15 due to the failure of the high frequency transmitter which would have maintained radio contact while the craft was out of range of the ultra-high frequency (UHF) receivers on the ground.

As a result of the problems with the craft, the second Soyuz module which was to have carried cosmonauts to perform an extra-vehicular activity (EVA) to the Soyuz 1 was not launched and the mission was cut short. Komarov was ordered to re-orient the craft using the ion system on orbits 15 to 17. The ion engine system failed. Komarov did not have enough time to attempt a manual re-entry until orbit 19. Manual orientation relied on using the equipped Vzor device, but in order to do this, Komarov needed to be able to see the Sun. To reach the designated landing site at Orsk the retro-fire would need to take place on the night side of the Earth. Komarov oriented the spacecraft manually on the dayside then used the gyro-platform as a reference so that he could orient the craft for a night side retro-fire.[27] He successfully re-entered the Earth’s atmosphere on his 19th orbit, but the module’s drogue and main braking parachute failed to deploy correctly and the module crashed into the ground, killing Vladimir Komarov. According to the 1998 book Starman, by Jamie Doran and Piers Bizony, as Komarov sped towards his death, U.S. listening posts in Turkey picked up transmissions of him crying in rage, “cursing the people who had put him inside a botched spaceship.”