H3-subtype influenza viruses are known to infect avian and mammalian species, including humans. However, little is known about the prevalence of H3 influenza virus infection in chicken populations in China. Therefore, a serologic survey of chickens was conducted in China to investigate the seroprevalence of avian H3-subtype influenza virus. Anti-H3 antibodies were assayed by using hemagglutination inhibition (HI) and confirmatory virus neutralization (VN) testing of 4598 serum samples, collected between July 2006 and June 2007, from 173 chicken flocks located in 18 areas that included 16 provinces and two municipalities. Seroepidemiologic results indicated that avian H3-subtype viruses were circulating in chickens in some regions of China, regions that included 12 of the 18 test areas, with an overall average prevalence rate of 2.83%. Samples from 44 of 173 flocks were HI/VN seropositive, including 15 flocks with levels that ranged from 10.00% to 41.94%. Significantly higher seroprevalence rates were observed in older chicken flocks and in those sampled in the cooler seasons. Standardized comparisons showed that Guangdong and Jiangsu, located in the south and east of China, respectively, had significantly higher levels of H3 seropositivity. For the first time, these results demonstrated serologic evidence for H3 avian influenza virus infection in chicken populations in several locations throughout China. These observations highlight the need for continued epidemiologic surveillance of the H3 subtype and for other low-pathogenic avian influenza viruses in China and other regions.