We identified females of six species of leafhoppers (tribe Empoascini), Asymmetrasca decedens (Paoli), Empoasca affinis Nast, E. decipiens Paoli, E. pteridis (Dahlbom), E. vitis Göthe, and Jacobiasca lybica (Bergevin and Zanon), using morphological characters of the ovipositor and isozyme and mitochondrial COI sequence analyses. Useful traits of the ovipositor are tooth morphology, length of the toothed area, form of sculpturing, and number and position of sensilla on the first and second pair of valves. Among the set of enzyme systems assayed, four putative loci, sod, hk-2, est-3, and est-2, together provided reliable diagnostic characters for single species, pairs of species, and groups of species. Analysis of mtCOI sequences revealed differences between the six species of Empoascini, and the neighbour-joining tree separated them into three distinct groups. Phenetic trees elaborated using Nei's index and the Kimura two-parameter model clustered the six Empoascini species in similar groups.