The Peromyscus maniculatus species complex is one of the most widespread group of small mammals in North America. However, the taxonomy and phylogenetic relationships among its constituent taxa remain unclear. As part of a revision of Peromyscus specimens from the highlands of the Trans-Mexican Volcanic Belt in central Mexico, we identified five individuals collected in 1968 that differed externally from other Peromyscus specimens, although morphologically similar to P. labecula and P. melanotis, both latter in the P. maniculatus species complex. Based on cranial measurements and mitochondrial DNA sequences, we aimed to more accurately determine the phylogenetic relationships and the taxonomic status of these individuals. Molecular phylogenetic analyses showed that the specimens formed a monophyletic clade sister to the P. maniculatus species complex. Pairwise genetic distances between those specimens and other species within the P. maniculatus species complex were greater than 7.91%. In addition, morphological analyses clearly distinguished the test specimens from P. melanotis and P. labecula. Based on the results of our molecular and morphological analyses, we conclude that these specimens represent an undescribed species of the P. maniculatus species complex, which we describe herein.