Oligoryzomys is a widespread and speciose genus of Neotropical rodents of the subfamily Sigmodontinae, including several species that are natural reservoirs of hantavirus and other zoonotic pathogens. Although Sigmodontinae species are generally considered to be species-specific reservoirs of Orthohantavirus, Oligoryzomys microtis is an exception, as it is the reservoir of four different genotypes of the Rio Mamoré orthohantavirus: RIOMV, HTN-007, RIOMV-3, and RIOMV-4. Here we demonstrate, based on comparative morphology, karyology, and phylogenetic analyses of the mitochondrial gene cytochrome b and the intron 7 of the β-fibrinogen nuclear gene, that O. microtis is a cryptic species complex with three geographically structured lineages and two different karyotypes. The lineages are parapatric, with one occurring in western Amazonia and north of the Amazonas-Solimões River, the second in transitional areas (ecotones) from central Amazonia with the Cerrado of central Brazil, and the third in eastern Amazonia south of Amazonas River. We establish that one lineage of O. microtis is the reservoir of the Rio Mamoré orthohantavirus genotypes RIOMV, HTN-007, and RIOMV-3 from Peru, Bolivia, and the Brazilian states of Amazonas and Acre. The second lineage is the reservoir of orthohantavirus Rio Mamoré genotype RIOMV-4 from the Brazilian state of Rondônia, whereas no orthohantavirus has been associated with the third lineage. The second and third lineages differ from O. microtis sensu stricto in chromosomal fundamental numbers (64 versus 66). The three lineages form monophyletic units in the cytochrome b tree, each supported by several synapomorphies. However, the three forms are morphologically very similar regarding external, cranial, and dental traits. Based on these karyological, molecular, and morphological results, we describe a new species of Oligoryzomys with two subspecies, and discuss the implications of our taxonomic revision for the evolutionary history of Rio Mamoré orthohantavirus.