"Poultry deaths at the farm were caused by a highly virulent strain of the H5N8 virus," the agriculture ministry said.
On Thursday, a suspected AI case was reported at the farm in Gochang, some 300 kilometers southwest of Seoul in North Jeolla Province.
Provincial quarantine authorities culled more than 20,000 ducks Friday after a preliminary test indicated a possible AI outbreak.
The latest discovery is the first case since May 2011, when more than 3 million poultry were slaughtered.
Earlier in the day, another suspected case was reported from a duck farm in Buan, a county adjacent to Gochang, the ministry said.
Quarantine officials confirmed the deaths of some 90 ducks in an on-site probe, and the remaining ducks are also showing AI symptoms, according to the ministry, adding that a DNA test is underway.
But the case turned out to have no connection with Gochang as no trade had been made between the farms, the ministry said.
Determining the exact strain of AI is often crucial as highly pathogenic strains may require more severe quarantine measures than low pathogenic strains.
Confirmation of an AI outbreak is followed by the immediate launch of intensive quarantine measures that include a lockdown of all poultry and personnel at farms in affected areas.
The ministry said it has also began taking initial quarantine measures at 24 poultry farms in four provinces that have recently purchased ducks from the affected farm in Gochang.