Karyologic comparisons of Nectomys with 2n = 52 and 2n = 56, captured in different regions of Brazil, revealed the existence of two presumptive tandem fusions. These differences proved to represent drastic reproductive barriers between these two karyologic groups, based on an analysis of spermatogenesis in captive-bred hybrids (2n = 54). In these hybrid specimens, cytologic and histologic analyses indicated severe meiotic arrest and the presence of two trivalents in the first meiotic division, in which 26 elements were frequently observed. These findings and the sterility of crosses between F1 males and females as well as backcrosses indicated that Nectomys populations with 2n = 52 and 2n = 56 should be considered different species and that the present taxonomic arrangement of this genus must be reviewed.