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Bats from the genus Vampyressa Thomas, 1900 are known from a relatively small number of individuals in Mexico. Through recent collecting efforts in southwestern Mexico, we detected several previously unknown populations of Vampyressa , which appeared to represent a lineage independent from but closely related to V. thyone. Here, we describe this lineage as a new species of the genus Vampyressa from the Mexican states of Guerrero and Oaxaca. The new species is currently known from at least 8 collected specimens and 3 released individuals from 8 localities, and can be differentiated from V. pusilla and V. thyone based on pelage and skull characters, size, mitochondrial Cytochrome b, and nuclear Recombination-activating gene 2 sequence data. The new species has been captured mainly in the lowlands but occurs along a wide altitudinal range from 150 to 2,200 m above sea level. Most of the records of the new species are from west of the Isthmus of Tehuantepec, which evidences a new case of a phylogenetic break at this biogeographic barrier. The genus Vampyressa now includes 6 species, and Mexico expands its position as the country with the largest number of endemic bats in the Neotropics, totaling 20 endemics. [ABSTRACT FROM AUTHOR]