Trinomys Thomas (1921) is a terrestrial genus of spiny rats endemic to the Brazilian areas of Atlantic Forest and the transitional areas of Cerrado and Caatinga. Although most species have been already karyotyped, the available cytogenetic information is mostly restricted to diploid and fundamental numbers. We analyzed the chromosomes of two Trinomys species: Trinomys moojeni (2 n = 56, FN = 106) and Trinomys setosus setosus (2 n = 56, FN = 106 and 2 n = 56, FN = 108). Our analyses included GTG- and CBG-banding, silver-staining of the nucleolar organizer regions, and chromosome mapping of telomeres and 45S rDNA by fluorescent in situ hybridization (FISH). Comparative GTG- and CBG-banding suggested that the interspecific variation may be due to rearrangements such as pericentric inversions, centromere repositioning, and heterochromatin variation. We report two new karyotypes for T. s. setosus and describe for the first time the banding patterns of the two Trinomys species.