Morphological, molecular and chromosomal studies in the generaLonchothrixandMesomyshave contributed to a better understanding of taxonomic design, phylogenetic relationships and karyotypic patterns. Recent molecular investigations have shown a yet undescribed diversity, suggesting that these taxa are even more diverse than previously assumed. Furthermore, some authors have questioned the limits of geographic distribution in the Amazon region for the speciesM.hispidusandM.stimulax. In this sense, the current study sought to understand the karyotypic evolution and geographic limits of the genusMesomys, based on classical (G- and C-banding) and molecular cytogenetic analysis (FISH using rDNA 18S and telomeric probes) and through the sequencing of mitochondrial genes Cytochrome b (Cytb) and Cytochrome Oxidase—Subunit I (CO using phylogeny, species delimitation and time of divergence, from samples of different locations in the Brazilian Amazon. The speciesM.stimulaxandMesomyssp. presented 2n = 60/FN = 110, whileM.hispiduspresented 2n = 60/FN = 112, hitherto unpublished. Molecular dating showed thatMesomysdiversification occurred during the Plio-Pleistocene period, withM.occultusdiverging at around 5.1 Ma, followed byMesomyssp. (4.1 Ma) and, more recently, the separation betweenM.hispidusandM.stimulax(3.5 Ma). The ABGD and ASAP species delimiters support the formation of 7 and 8 potential species of the genusMesomys, respectively. Furthermore, in both analyzesMesomyssp. was recovered as a valid species. Our multidisciplinary approach involving karyotypic, molecular and biogeographic analysis is the first performed inMesomys, with the description of a new karyotype forM.hispidus, a new independent lineage for the genus and new distribution data forM.hispidusandM.stimulax.