An outbreak reported Wednesday at a farm around 300 kilometres (187 miles) southeast of Seoul was confirmed after blood tests as the virulent H5N1 strain of the disease, the agriculture ministry said.
It raised to 23 the total number of outbreaks reported across six provinces of South Korea.
Only the northeastern province of Gangweon, North Chungcheong in the centre and the southern island of Jeju have been unaffected so far, according to the ministry.
Meanwhile quarantine authorities denied allegations they had covered up an outbreak of avian influenza in the province of North Gyeongsang last month.
Responding to a television report, they said they had been cautious of the results of preliminary tests on poultry at Yeongcheon City, but final results confirmed the outbreak.
South Korea reported seven cases of H5N1 infection cases between November 2006 and March last year, resulting in temporary suspension of poultry exports to Japan, Hong Kong, Taiwan and elsewhere.
Last June, however, the World Organisation for Animal Health classified the country as free from the disease.
The H5N1 strain has killed more than 240 people worldwide since late 2003. No South Koreans are known to have contracted the disease.