West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.