Abstract: To investigate avian Tembusu virus infection in chickens,2 300sera samples were collected from highland laying hens and reserve hens in Fujian,Jiangxi,Zhejiang and Guangdong provinces during Dec.2010to Sep.2013. The antibodies were detected by indirect enzyme-linked immuosorbent assay(ELISA).The results showed that the samples of laying hens had higher seropositive rate(43.4%)than that of reserve hens(19.9%).For laying hens, specimens from Fujian province had the highest seropositivity(60.7%),and for reserve hens the specimens from Guangdong province had the highest seropositivity(37.2%).The antibodies for avian Tembusu virus were detected from serum samples in all four provinces with different antibody positive rate,and the samples of laying hens had higher seropositive rate than reserve hens.The serological investigation indicated that different degrees of avian Tembusu virus infection existed in all these regions,providing the scientific basis for preventing avian tembusu virus infection in chickens in these regions and even in China.