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Genetic studies in the highlands of Mexico have revealed extensive intraspecific differentiation and highly structured genetic variation in rodents and other taxa. One of the most important and complex systems in these highlands is the Trans-Mexican Volcanic Belt (TMVB), a biogeographic region located in central Mexico. Recent studies suggest strong structure and undiscovered diversity within the TMVB highlands, which indicates more study of endemic species is warranted. One of these species is the endemic Volcano Harvest Mouse Reithrodontomys chrysopsis, which inhabits small and fragmented patches at high altitudes. This mouse is poorly known, under-represented in field collections, and has been the subject of few studies of phylogenetics, taxonomy, and evolutionary history. This study aimed to assess the phylogenetic and intraspecific relationships of R. chrysopsis throughout its known geographic distribution to better understand the evolutionary patterns of it and other TMVB mammals. Our phylogenetic analyses using Cytb and Fgb-I7 markers revealed that R. chrysopsis is sister to R. sumichrasti and R. megalotis, which contrasts with a previous hypothesis based on morphological data. Genetic differentiation, divergence time, and species delimitation analyses showed three main divergent lineages within R. chrysopsis, located at the western, central, and eastern TMVB. These lineages diverged during the Pliocene-Pleistocene interval. Our results support the hypothesis that volcanic activity and climatic oscillations during the Pleistocene in central Mexico promoted diversification processes within the TMVB. Further molecular and morphological studies will be needed to provide information for the taxonomic designation of the east divergent lineage detected herein. [ABSTRACT FROM AUTHOR]