A government-funded research team from Seoul National University found the footage, which was filmed in 1944 by an American soldier, in the United States National Archives after a two-year hunt.
"South Korea has not had its own data on comfort women and (has) been relying on Japanese and American data," said Kang Sung-hyun, an academic on the team.
"It is crucial for South Korea to have our own data for the issue of comfort women," he said.
The 18-second black-and-white clip shows seven women lined up outside a brick house, being questioned by Chinese soldiers. The women were found by US-China allied forces in China's Yunnan province, the research team said in a statement.
The women were registered by US soldiers, the research team said. Two of the women in the video had already appeared in previously released photos of "comfort women".
The term is used to describe girls and women from South Korea, China, the Philippines and elsewhere, who were forced into prostitution in Japanese wartime military brothels.
South Korean activists estimate that there may have been as many as 200,000 Korean victims.
However, the issue continues to strain relations between the two countries.
Japan wants South Korea to remove a statue near the Japanese consulate in Busan city commemorating Korean comfort women, as well as another near the Japanese embassy in Seoul, saying that the presence of the statues violates the 2015 agreement.
South Korean President Moon Jae-in suggested during his campaign for a May 9 election that most South Koreans did not accept the 2015 deal negotiated by his conservative predecessor Park Geun-hye and Japanese PM Shinzo Abe, and that he could try to renegotiate it.
Since the election, he has not directly addressed whether he will seek to renegotiate the pact. The issue is likely to be discussed when he meets Abe on Friday on the sidelines of the Group of 20 summit in Hamburg, Germany.
($1 = 113.1500 yen)