2008. Heterogeneity of heterochromatin in six species of Ctenomys (Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed by a combined analysis of C-and RE-banding. Acta Theriologica 53: 57-71.Exceptional chromosomal variability makes Ctenomys an excellent model for evolutionary cytogenetic analysis. Six species belonging to three evolutionary lineages were studied by means of restriction endonuclease and C-chromosome banding. The resulting banding patterns were used for comparative analysis of heterochromatin distribution on chromosomes. This combined analysis allowed intra-and inter-specific heterochromatin variability to be detected, groups of species belonging to different lineages to be characterized, and phylogenetic relationships hypothesized from other data to be supported. The "ancestral group", Ctenomys pundti and C. talarum, share three types of heterochromatin, the most abundant of which was also found in C. aff. C. opimus, suggesting that the latter species also belongs to the "ancestral group". Additionally, within the subspecies C. t. talarum, putative chromosomal rearrangements distinguishing two of the three chromosomal races were identified. Two species belong to an "eastern lineage", C. osvaldoreigi and C. rosendopascuali, and share only one type of heterochromatin homogeneously distributed across their karyotypes. C. latro, the only analyzed species from the "chacoan" lineage, showed three types of heterochromatin, one of them being that which characterizes the "eastern lineage". C. aff. C. opimus, because of its low heterochromatin content, is the most primitive karyotype of the genus yet described. The heterochromatin variability showed by these species, reflecting the evolutionary divergence toward different heterochromatin types, may have diverged since the origin of the genus. Heterochromatin amplification is proposed as a trend within Ctenomys, occurring independently of chromosomal change in diploid numbers.