AbstractIn this study, we describe a new Lampyridae subfamily, Chespiritoinae new subfamily, its sole genus Chespirito new genus, and three new species: Chespirito zaragozai new species, Chespirito lloydi new species, and Chespirito ballantyneae new species from the Neotropical portions of Mexico. Chespirito can be readily separated from all other known Lampyridae by the unique prosternum, characterized by being very wide, divided in the middle by a distinct suture forming two plates, with the anterior margin bearing a narrow inter-coxal process, the strongly setose filiform antennae, with antennomere III much smaller than all other antennomeres, the pronotum medially constricted (not in C. ballantyneae), with area adjacent to disc strongly punctate and with the presence of a strongly developed longitudinal carina. To test the placement of the genus within the Lampyridae, we obtained three genetic markers (18S rRNA, 28SrRNA, and cox1 mitochondrial DNA) from C. zaragozai and performed a maximum likelihood (ML) and Bayesian inference (BI) analysis. Our analyses rendered nearly identical tree topologies, with C. zaragozai new species recovered as an independent lineage as sister to Pollaclasis bifaria (Say) (Coleoptera, Lampyridae) + Cyphonocerus ruficollis Kiesenwetter (Coleoptera, Lampyridae)+Luciolinae, with a posterior probability of 96 for the BI analysis and UFBoot respectively of 91 for the ML analysis with the entire clade sister to Pterotus obscuripennis LeConte (Coleoptera, Lampyridae). A detailed examination of the morphology of Chespirito indicates that this lineage is divergent from all other known Lampyridae, which combined with the results of our analyses supports the erection of a new subfamily.