AbstractA data set comprising DNA sequences from 388 loci and >99,000 aligned nucleotide positions, generated using anchored hybrid enrichment, was used to estimate relationships among 138 leafhoppers and treehoppers representative of all major lineages of Membracoidea, the most diverse superfamily of hemipteran insects. Phylogenetic analysis of the concatenated nucleotide sequence data set using maximum likelihood produced a tree with most branches receiving high support. A separate coalescent gene tree analysis of the same data generally recovered the same strongly supported clades but was less well resolved overall. Several nodes pertaining to relationships among leafhopper subfamilies currently recognized based on morphological criteria were separated by short internodes and received low support. Although various higher taxa were corroborated with improved branch support, relationships among some major lineages of Membracoidea are only somewhat more resolved than previously published phylogenies based on single gene regions or morphology. In agreement with previous studies, the present results indicate that leafhoppers (Cicadellidae) are paraphyletic with respect to the three recognized families of treehoppers (Aetalionidae, Melizoderidae, and Membracidae). Divergence time estimates indicate that most of the poorly resolved divergence events among major leafhopper lineages occurred during the lower to middle Cretaceous and that most modern leafhopper subfamilies, as well as the lineage comprising the three recognized families of treehoppers, also arose during the Cretaceous.