A recent systematic revision of the mayfly genus Ephemerella by Jacobus and McCafferty (2003) synonymized 9 species having similar morphology but distinct geographic ranges and different life history traits into a single, broadly distributed species, Ephemerella invaria. Strong population genetic structure has been found to occur even at small spatial scales in mayflies, suggesting that reproductive isolation among geographically distinct populations may be common in this taxon. I conducted a molecular analysis using the COI subunit of mitochondrial DNA to see whether genetic relationships among closely-related Ephemerella species were more concordant with the groupings based on ecological or morphological traits. Parsimony and maximum likelihood analyses produced similar trees in which E.invaria and E.inconstans were monophyletic, but other species, including the invaria synonyms E. floripara and E. rotunda, were unresolved or problematic. The results suggest a complex pattern of colonization and geographic differentiation, supporting elements of both the historical and revised systematics of this genus.