The occurrence of species is inadequately known for Morelos, one of the smallest states at the center of Mexico. Our research documented the occurrence of ant species in the State. Specimens were collected in four conserved areas and a range of disturbed habitats. Morphological traits and molecular sequences of the Cytochrome Oxidase subunit I (COI) were used as complementary tools to identify species. We identified 164 ant species of which 48 were new records for the State. Molecular analysis helped confirm and document the identity of species in groups of ants. The study could be used as a model to assess ecosystem health across extensive geographical regions.