The genus Ctenomys (Rodentia: Ctenomyidae) shows several taxonomic inconsistencies. In this study, we used an integrative approach including DNA sequences, karyotypes, and geometric morphometrics to evaluate the taxonomic validity of a nominal species, Ctenomys bicolor, which was described based on only one specimen in 1912 by Miranda Ribeiro, and since then neglected. We sampled near the type locality assigned to this species and collected 10 specimens. A total of 820 base pairs of the cytochrome b gene were sequenced and analyzed together with nine other species and four morphotypes obtained from GenBank. Bayesian analyses showed that C. bicolor is monophyletic and related to the Bolivian-Matogrossense group, a clade that originated about 3 mya. We compared the cranial shape through morphometric geometrics of C.