Abstract The spiny rat Trinomys, with ten endemic species in eastern Brazil, has a complex taxonomy. We carried out a revision of the karyotypes of Trinomys, described for the first time the karyotype of Trinomys paratus and performed the first phylogenetic analysis including all Trinomys species based on the mitochondrial cytochrome b gene. The T. paratus karyotype showed diploid number of 58 and fundamental autosomal number of 112. Diploid and fundamental autosomal numbers (FNa), and chromosomes’ morphology, are similar to those described for Trinomys eliasi. T. paratus appears as sister taxa of T. eliasi; in turn, this clade was recovered as the sister group of Trinomys setosus, as previously reported, confirming that sister species of Trinomys has conserved karyotypes, and suggesting that karyological evolution in this genus could be slower than species differentiation.