The South American rodent Graomys griseoflavus is a species with a Robertsonian (Rb) autosomal polymorphism. A marked genetic differentiation between 2n = 42–41 and 2n = 38–34 karyomorphic groups was evidenced by cytogenetic and molecular analysis. The mitochondrial control region was sequenced in all Graomys karyomorphs for its characterization and used to trace more accurate phylogenetic relationships. The molecular organization showed to be coincident with the consensus molecular structure described for other rodent taxa, exhibiting the conserved domains ETAS (extended termination-associated sequences), CD (central domain) and CSB (conserved sequence block) 1, 2 and 3. Phylogenetic trees showed that 2n = 42–41 and 2n = 38–34 karyomorphic groups form separate clades, with neither phylogeographical structure nor population subdivision within Rb karyomorphs. These findings suggest a short evolutionary time for the occurrence and fixation of the chromosomal rearrangements and reinforce the single origin hypothesis for the Rb karyomorphs of G. griseoflavus.