Objective: To investigate serologically the presence of avian influenza virus (AIV) in backyard chickens from Mandlhakazi district, South Mozambique. Methods: A total of 439 sera samples from unvaccinated and apparently healthy backyard chickens from 4 villages (Chidenguele, Macuacua, Chizavane, and Nwadja-hane) were tested for the presence of AIV antibodies through commercial enzyme-linked immunoabsorbent assay (ELISA) kit used according to manufacturer instructions. Results: Anti-AIV antibodies were detected in all villages surveyed. The overall sero-prevalence obtained was 32.6％ (95％ CI 28.2％–37.0％). The highest prevalence of 51.3％ (95％ CI 42.3％–60.2％) was recorded in Macuacua village, while the lowest prevalence of 13.0％(95％CI 6.2％–19.9％) was found in Chizavane village. The results of logistic regression analyses suggested that chicken being located in Chizavane and Macuacua villages were more unlikely for getting the virus exposure (P<0.05). Conclusions: Our findings suggested that AIV is widespread within backyard chickens in the studied villages. Further research is needed to identify the circulating virus ge-notypes and determine the potential role of backyard chickens in the zoonotic trans-mission of AIV in Mozambique.