Filmed by US troops in China, the clip was found by government-funded researchers at Seoul National University in US archives.
The 18-second clip shows several women lined up talking to a Chinese soldier.
South Korean activists estimate 200,000 women were forced into brothels for Japan's military.
They are believed to have been mainly from Korea, but also from China, Indonesia, the Philippines, and Taiwan.
Until now, the only records of women forced into sexual slavery by the Japanese military during World War Two had been photographs and survivor testimonials.
The research team says the footage was filmed by joint US-Chinese troops in China's Yunnan province which was previously occupied by Japan.
The seven Korean women were freed in 1944 and the officer speaking with them has been identified as a Chinese captain of the China-US Combined Force, the team said.
The issue has long strained the relationship between South Korea and Japan over a perceived lack of adequate apology and compensation from Japan.
In 2015, the two countries reached a settlement whereby Tokyo formally apologised and agreed to pay 1bn yen ($8.3m, Â£5.6m) to fund victims.
Many Koreans though viewed the apology as insufficient and the issue continues to plague ties.
In January, Japan temporarily withdrew its ambassador to South Korea over a "comfort women" statue placed outside the Japanese consulate in Busan.
A similar statue has also been placed outside Japan's consulate in Seoul, and Tokyo wants both these statues to be removed.
South Korea has released what it says is the first known footage of "comfort women" forced to work as sex slaves for Japanese soldiers during World War Two.