Lytopylus is species-rich genus of Agathidinae (Hymenoptera: Braconidae: Agathidinae), but limited information of the genus has been published. Morphological, molecular, and ecological data were compared to resolve the species limits of Lytopylus reared from caterpillars collected the Area de Conservación Guanacaste (ACG) in Costa Rica. Molecular data were initially used to hypothesize species limits, and morphological and host use data were employed to make a final decision when molecular data was indecisive, e.g., when the genetic difference between species was slight. Thirty-two new species are described with image plates of each species. Phylogenetic analyses of the barcode region of the cytochrome c oxidase subunit I (COI) mitochondrial gene was conducted using Neighbor-Joining (NJ) and the Maximum likelihood (ML) analysis. A concatenated COI+28S dataset was analyzed by ML analysis to elucidate evolutionary patterns in ecological characters.