The taxonomic identification of Lonchothrix emiliae (Rodentia, Echimyidae, Eumysopinae) is problematic because of the overlap of morphological characters with its sister clade represented by species in the genus Mesomys which, like L. emiliae, is distributed throughout the Amazonian biome. Cytogenetic studies reported the karyotype of L. emiliae as 2n = 60/FN = 116, but this karyotype and samples were later designated as M. hispidus. To evaluate the karyotype diversity of Lonchothrix and Mesomys, and to provide data useful as karyological diagnostic characters, in the present study we made a comparative analysis of specimens of L. emiliae and M. stimulax collected from two Brazilian Amazonian localities, using C-banding, G-banding, FISH using rDNA 45S and telomeric probes, and Cytochrome-b (Cytb) sequences. The results indicate that L. emiliae has 2n = 64♀, 65♂/FN = 124 and a multiple sexual system (XX/XY1Y2), while M. stimulax has 2n = 60/FN = 116. The Neo-X system found in L. emiliae also occurs in two Proechimys species, but cytogenetic analysis indicated an independent origin for these systems. The rDNA 45S analysis showed interstitial signals at one autosomal pair for each species, while an ITS found in L. emiliae was not coincident with the NOR. The molecular analysis confirmed Lonchothrix and Mesomys are sister genera, and the high level of intraspecific genetic divergence (7.1%) in M. stimulax suggests that it may be a species complex.