DNA barcoding, using short DNA sequences from a standardized region of the genome, has been proposed as a tool for species identification and discovery. This study demonstrates that cytochrome c oxidase I (COI) DNA barcodes effectively distinguish species in three Lepidoptera families (Hesperiidae, Sphingidae, and Saturniidae) in the species-rich fauna of Area de Conservación Guanacaste, Costa Rica. Of the 521 species recognized, 97.9% had distinctive COI barcodes, with only a few instances of interspecific sequence overlap involving very similar species. Two or more barcode clusters were found within each of 13 supposedly single species, indicating overlooked species complexes. The results suggest that DNA barcoding will significantly aid species identification and discovery in tropical settings. COI barcodes identified all but 2.1% of the species in the study, with cases of incomplete resolution involving pairs or triplets of closely related species. The study also found that barcode clusters correlated with morphological and ecological traits, indicating that some species may be complexes of closely related species. DNA barcoding is effective for species recognition in tropical settings and can reveal overlooked species, even in well-studied groups. The study highlights the potential of DNA barcoding as a tool for species identification and discovery in biodiversity-rich areas.DNA barcoding, using short DNA sequences from a standardized region of the genome, has been proposed as a tool for species identification and discovery. This study demonstrates that cytochrome c oxidase I (COI) DNA barcodes effectively distinguish species in three Lepidoptera families (Hesperiidae, Sphingidae, and Saturniidae) in the species-rich fauna of Area de Conservación Guanacaste, Costa Rica. Of the 521 species recognized, 97.9% had distinctive COI barcodes, with only a few instances of interspecific sequence overlap involving very similar species. Two or more barcode clusters were found within each of 13 supposedly single species, indicating overlooked species complexes. The results suggest that DNA barcoding will significantly aid species identification and discovery in tropical settings. COI barcodes identified all but 2.1% of the species in the study, with cases of incomplete resolution involving pairs or triplets of closely related species. The study also found that barcode clusters correlated with morphological and ecological traits, indicating that some species may be complexes of closely related species. DNA barcoding is effective for species recognition in tropical settings and can reveal overlooked species, even in well-studied groups. The study highlights the potential of DNA barcoding as a tool for species identification and discovery in biodiversity-rich areas.