The second round will be between two Members of the European parliament, Yannick Jadot, 49, who won 35.61 percent, and Michèle Rivasi, 63, who won 30.16 percent.
The only candidate beaten by Duflot was Karima Delli, 37, who won 9.82 percent.
Duflot said she was "disappointed" by the result on her Facebook page and declined to declare support for either of the two remaining candidates.
The defeat was all the more humiliating in that Rivasi, a former Socialist who was once head of Greenpeace France who joined EELV in 2005, was a surprise candidate, who had to lobby hard at the party's summer school to collect the necessary 36 nominations that allowed her to take part in the primary.
Only 17,148 people registered to take part in the primary - 7,000 of them being EELV members.
Splits and decline in support
The party has been weakened by splits of several MPs who opposed Duflot's decision to quit the government and claim it has become "sectarian".
One of them, Jean-Vincent Placé, who is currently junior minister in charge of governmental reform, described the primary result as "a form of collective suicide".
Another, Barbara Pompili, now a junior minister in charge of biodiversity, blamed Duflot for the party's decline.
"Because of her political ecology is considerably weakened," she commented. "And when you have a strategy of isolation, as she did, you end up alone."
The party's national conference in June rejected all alliances with the Socialist Party.
Campaigning without hoping to win
Duflot, the only possible candidate to have figured in opinion polls up until now, was at two to three percent but still campaigned on the claim that she could become the first ecologist president.
Jadot has said that "nobody seriously believes today that the ecologists can win the 2017 presidential election".
He, too, once held a position in Greenpeace France and, although he is reported to have had differences with Rivasi there, may have difficulty differentiating himself from her due to their similar careers.