The neotropical rodents Akodon cursor (2n = 14, 15, and 16) and A. montensis (2n = 24 and 25), two closely related and morphologically indistinguishable species, have been compared by G-banding and chromosome painting. In situ hybridization of a biotinylated DOP-PCR product obtained from a microdissected chromosome of A. cursor onto A. montensis chromosomes was performed in combination with localization of telomeric sequences using a (TTAGGG)n oligomer as a FISH probe. The results provide evidence of the complex chromosomal rearrangements suggested by GTG-banding analysis, indicating the origin of one A. cursor autosome from three different A. montensis autosomes. Furthermore, the complete cytogenetic homeology between the A. cursor and A. montensis karyotypes was determined, along with the occurrence of tandem fusions and pericentric inversions and the loss of telomeres, centromeres, and chromosome arms. Evidence for the ancestral origin of the A. cursor karyotype is also provided.