This study represents the first comprehensive total evidence analysis for pannote mayflies with emphasis on the family Ephemerellidae. A robust phylogeny was constructed based on approximately 250 morphological characters combined with molecular data. DNA sequence data were generated from the following genes: 18S rDNA, 28S rDNA, 12S rDNA, 16S rDNA, and Histone3. Taxon sampling includes 35 exemplars representing 17 of the 19 described ephemerellid genera. Specifically, the monophyly of the family Ephemerellidae was tested and the major lineages (subfamilies) within the family were identified. The position of Ephemerellidae in relation to other families within the superfamily Ephemerelloidea was clarified. The evolution of body armature, eyes, and gills for Ephemerellidae will be discussed in light of the topology.