A sample of 101 specimens of Ctenomys minutus was collected along its geographic range. Eight karyotypes (2n = 42, 45, 46a, 46b, 47, 48, 49 and 50) were found. The chromosome polymorphisms were due to Robertsonian rearrangements and tandem fusions. The distribution of polymorphisms indicated three population blocks: northern (2n = 49 and 50), central (2n = 46a, 47, and 48) and southern (2n = 42, 45, and 46b). These findings suggest that this species is undergoing a speciation process due to geographic isolation.