BackgroundThe hemipteran insect family Cicadellidae (leafhoppers) includes >2,600 valid genera and >22,000 valid species worldwide, including >2,000 species in China. Typhlocybinae, second largest subfamilies of Cicadellidae, is widely distributed in the six major zoogeographic regions of the world, including >4,000 species worldwide and >1,000 species in China. Previously, morphological analysis are often effective to the way of taxonomy, but it did not combine with molecular biology. Therefore, morphology and mitochondrial genomes (mitogenomes) of two leafhopper species, Eupteryx (Eupteryx) minuscula Lindberg, 1929 and Eupteryx (Stacla) gracilirama Hou, Zhang & Huang, 2016 were studied and analyzed. This study analyzed the morphological and molecular characteristics of the two leafhoppers, and showed whether the results of the two identifications were consistent.MethodsBased on the method of comparison, mitogenomes and morphology were analyzed to prove the relationship between the two leafhoppers.ResultsAlthough two focal species are classified in two different subgenera of the same genus, they still share many morphological features, such as the moderately produced crown fore margin; the milky yellow apical part of scutellum; the pronotum, basal triangles of scutellum, and forewing are dark with several colorless patches on the surface; the light yellow face, without any spots or stripes, and so on. The circular mitogenomes are 16,944 bp long in E. minuscula (GenBank: MN910279) and 17,173 bp long in E. gracilirama (GenBank: MT594485). All of the protein-coding genes are starting with ATN, except for some in mitogenome, which has a single T or TAN as a stop codon. All tRNAs have the typical cloverleaf-shaped structure except for trnS1 (AGN) (E. minuscula) which has a reduced DHU arm. Moreover, these two mitogenomes have trnR with an unpaired base in the acceptor stem. The phylogenetic relationships between E. minuscula and E. gracilirama in respect to related lineages were reconstructed using Maximum likelihood and Maximum parsimony analyses.DiscussionThe result showed that the tribe Typhlocybini is a sister to the tribes Erythroneurini and Empoascini, and five genera, Bolanusoides, Typhlocyba, Eupteryx, Zyginella and Limassolla are forming a single clade. E. minuscula and E. gracilirama are clustered together, supporting the monophyly of the genus Eupteryx. The above conclusions are consistent with the traditional classification of the subfamily.