Abstract The Heteromys pictus–spectabilis species complex currently consists of two species: H. pictus, which is distributed across western, central, and southern Mexico; and H. spectabilis, an endangered species which is restricted to a small region of southeastern Jalisco. Previous phylogenetic studies have indicated that H. spectabilis renders H. pictus as paraphyletic, and therefore revisions are needed to resolve this paraphyly. Phylogenetic trees based on mitochondrial DNA and nuclear DNA genes were constructed using maximum likelihood and Bayesian methods, grouping individuals into previously established Cytochrome b (Cytb) haplogroups across the geography. Phylogenetic trees were run through bPTP and GMYC analyses to estimate the number of predicted species that may be present, and when these lineages likely diverged based on the genes tested. To assist with species delimitation predictions (based on the Genetic Species Concept), intra- and interspecific Kimura two-parameter (K2P) values were calculated to predict species-level lineages within the species complex. We found evidence to support the hypothesis that there are multiple cryptic species present within H. pictus, with some K2P values between the Cytb haplogroups being on par with what is expected between different species and genera within Rodentia. This is further supported by the phylogenetic trees (individual and concatenated) constructed from Cytb and three nuclear genes (Bfib, IRBP, and PRKCI), which consistently group certain Cytb haplogroups together in ways that correspond to geographic filter barriers in Mexico.