An annotated checklist consisting of 300 species is provided for the mayfly fauna of the southeastern United States [Alabama (139 spp.), Arkansas (118 spp.), Florida (77 spp.), Georgia (155 spp.), Kentucky (135 spp.), Louisiana (61 spp.), Mississippi (59 spp.), North Carolina (207 spp.), South Carolina (185 spp.), Tennessee (167 spp.), Virginia (170 spp.), West Virginia (119 spp.)]. The list is based on previously published and newly published collection data. Some 107 supported state records in the region are also provided based on 92 of the species. The Southeast is the richest region in North America in terms of mayfly biodiversity, but the states of Louisiana and Mississippi remain poorly known.