Five Imparfinis mirini and one Imparfinis minutus populations were studied using basic cytogenetic and molecular techniques. Cytogenetic analysis showed that I. mirini individuals presented the same diploid number 2n=58 (FN=116). However, they presented two distinct karyomorphs: karyomorph A (36m+18sm+4st) for the Mogi-Guaçu and Paranapanema basin populations, and karyomorph B (42m+12sm+4st) for the Tietê basin populations. I. minutus populations from the Paraíba do Sul basin presented a karyotype identical to karyomorph A of I. mirini. C-banding also presented distinct patterns, with a greater amount of heterochromatin, most of which was pericentromeric and interstitial for karyomorph A I. mirini and I. minutus. There was a minor amount of heterochromatin in karyomorph B, most of which was terminal and interstitial. Simple and interstitial nucleoli organizer regions were located in the biggest metacentric pair of the complement in all populations with GC-rich nature, and this location was confirmed by the fluorescent in situ hybridization technique with 18S ribosomal DNA with 5S rDNA synteny. In molecular analysis by DNA barcoding, two other populations from the Tietê basin were added. The phylogram showed that the populations were more related to the intrabasin. Cytogenetic resemblance among specimens from distinct basins may be the result of either recent convergence or ancestral feature retention not followed by mutations in mitochondrial DNA.