Partial 18s rDNA sequences from 22 exemplar mayfly species (Ephemeroptera) representing 20 families were analyzed to obtain a best phylogenetic tree for comparison to previous phylogenetic hypotheses. With respect to relationships among the three major groupings, our molecular data support the hypothesis that Pisciforma and Setisura comprise a monophyletic sister group to the Furcatergalia, rather than the hypothesis that Setisura and Furcatergalia comprise a monophyletic group stemming from the Pisciforma. Within Pisciforma, acceptable trees show that Baetidae separates at the base of the Pisciforma clade. The data suggest that Pisciforma is paraphyletic and do not support the grouping of all Southern hemisphere families as a monophyletic group. An evolutionary sequence favored by the data suggests a grouping of Siphlonuridae, Rallidentidae, Nesameletidae, and Ameletidae and a grouping of Oniscigastridae, Ameletopsidae, and Acanthametropodidae. The data support the monophyly of Setisura (Heptageniidae, Arthropleidae, Pseudironidae, Oligoneuriidae, Isonychiidae, and Coloburiscidae). Within Setisura, a bootstrap/jackknife test places the families Heptageniidae, Arthropleidae, and Pseudironidae in one clade at 100% frequency. Also supported are hypotheses that Pseudironidae is a sister group to a Heptageniidae–Arthropleidae group and that a sister relationship exists between the latter two families. Hypotheses that Pseudironidae separated from other Setisura families at an earlier stage and comprises a sister group to a Heptageniidae–Oligonuriidae lineage or that Pseudironidae should be moved out of Setisura are not supported.