Its wide karyotypic variation characterizes the genus Ctenomys, and in Brazil, the genus is distributed in the country's southern, Midwest, and Northern regions. Recently, populations of Ctenomys have been found in the Midwest and northern Brazil, with two new lineages named C. sp. "xingu" and C. sp. "central". This work combines classical cytogenetic and molecular analyses to provide new chromosomal information on the boliviensis group distributed in Northern and Midwestern Brazil. This includes the validation of the karyotype of C. bicolor and C. nattereri, and the description of the karyotype of C. sp. "xingu" and C. sp. "central". We found three different karyotypes: 2n=40 for C. bicolor; 2n= 36 for C. nattereri, and specimens from a locality belonging to C. sp. "central"; 2n=34 for the lineage C. sp. "xingu" and specimens from a locality belonging to C. sp. "central". Furthermore, GTG-banding revealed homologous chromosomes between species/lineages and allowed the identification of the rearrangements that occurred, which proved the occurrence of fissions. Considering our results on the variation of 2n in the boliviensis group, we found two possibilities: the first, deduced by parsimony, is that 2n=36 appeared initially, and two fissions produced gave rise to 2n=40, and an independent fusion gave rise to 2n=34 from 2n=36; moreover, the second explanation is that all karyotypes arose independently.
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