The former Southeast Asian Games champion unexpectedly lost 0-1 to Chinese Taipei at the Mansalarthiri Stadium, Mandalay City, Myanmar.
Coach Norimatsu Takashi used his best squad against a rival who was considered the weakest in the Group A and his team created the first chance.
In the 17th minute, Veteran striker Nguyen Thi Minh Nguyet reached close to goalkeeper Tsai Ming-Jung of the other side but her shot went a little wide.
Vietnamese players easily set up their domination and found a number of opportunities which, however, could not be converted. Nguyet Nguyen Hai Hoa and Tran Thi Thuy Trang all failed to enter Tsai's net.
Taipei's defenders successfully resisted Vietnam's consecutive attacks until the interval.
Takashi retained the same squad in the second half using the 4-5-1 formation against Taipei who spent time training hard in Hanoi last month.
Their improved skills, better height and physique, were brought into play in this half of the match.
Goalie Dang Thi Kieu Trinh of Vietnam had to work hard with strong support from her defenders to save her net.
However that could only last until the 85th minute.
In a rapid counter-attack, substitute defender Lin Chiung Ying received a ball from the right wing and her straight shot to Trinh's far corner made the opening goal.
With five minutes remaining, Vietnam made desperate attempts to score, but could not do much to change the score line.
The loss has made things difficult for Vietnam en route to the final qualification round.
In a press-briefing prior to the match, Takashi, who travelled with his players to his home country in Japan for an intensive training course, set a target of staying on top of the group to take the only ticket to advance.
In their preparation for the tournament, Vietnam played Taipei twice in July. They won one and drew one.
On September 18, Vietnam will play host Myanmar who beat Jordan 2-0 in another match yesterday.
It is said to be a hard game for both sides who have played each other on numerous occasions in regional and SEA Games events.
In their latest meet, Vietnam won 3-2 in the ASEAN Football Federation Women's Championship on their home turf of Thong Nhat Stadium in HCM City on May 2.
The second qualifying round of the 2016 Olympic Games-Asian Zone's Group A sees five participants. Teams will compete in a round-robin event from September 14-22.
The best team will join the Republic of Korea, China, and North Korea, in addition to Australia and hosts Japan in the last qualifier, which is scheduled from February 29 to March 9.
Two outstanding squads from this round will book their berths to the Rio de Janeiro Games next summer.
