The geographical distribution and impact on animal and human health of both West Nile and Usutu viruses, two flaviviruses of the Japanese encephalitis complex, have been increasing during the past two decades. Both viruses circulate in Europe and Africa within a natural cycle between wild birds and mosquitoes, mainly from the Culex genus. We retrospectively analyzed sera from domestic and wild birds sampled in 2008 in two wetlands, namely the Inner Niger Delta, Mali, and the Lake Alaotra area, Madagascar. Sera were first tested using a commercial ID Screen West Nile Competition Multi-species ELISA kit. Then, positive sera and sera with insufficient volume for testing with ELISA were tested with a Microneutralization Test. In Mali, the observed seroprevalence in domestic birds was 28.5% [24.5; 32.8] 95%CI, 3.1 % [1.8; 5.2] 95%CI, 6.2% [3.4; 10.2] 95%CI and 9.8 % [7.3; 12.8] 95%CI, for West Nile virus (WNV), Usutu virus (USUV), undetermined flavivirus, and WNV/USUV respectively. Regarding domestic birds of Madagascar, the observed seroprevalence was 4.4 % [2.1; 7.9]95%CI for WNV, 0.9% [0.1; 3.1] 95%CI for USUV, 1.3% [0.5; 2.8] 95%CI for undetermined flavivirus, and null for WNV/USUV. Among the 150 wild birds sampled in Madagascar, two fulvous whistling-ducks (Dendrocygna bicolor) were positive for WNV and two for an undetermined flavivirus. One white-faced whistling-duck (Dendrocygna viduata) and one Hottentot teal (Spatula hottentota) were tested positive for USUV. African and European wetlands are linked by wild bird migrations. This first detection of USUV—as well as the confirmed circulation of WNV in domestic birds of two wetlands of Mali and Madagascar—emphasizes the need to improve the surveillance, knowledge of epidemiological patterns, and phylogenetic characteristics of flavivirus in Africa, particularly in areas prone to sustained, intense flavivirus transmission such as wetlands.