In this study, we conducted a survey of newly discovered populations of tuco-tuco (subterranean rodents of the genus Ctenomys) in the Entre Ríos province, in an area characterized by its unexplored nature and its climatic and biogeographic complexity within Argentina, which includes two National Parks. We characterize the nucleotide sequences of the cytochrome-b gene, revealing the presence of seven novel haplotypes within Ctenomys rionegrensis, a species known to inhabit both sides of the Uruguay River. Through Bayesian analyses, we estimated the divergence times of the oldest lineages of C. rionegrensis, as well as those of the haplotypes located east of the Uruguay River, dating back approximately 630,000 years before present (ybp) and 526,000 ybp, respectively. These estimates correspond with significant paleogeographic events in the region. Our findings may raise questions regarding the taxonomic classification of the species and suggest potential modifications to its current endangered status as designated by the International Union for Conservation of Nature (IUCN). Consequently, this research provides valuable insights that may inform future revisions of the species' conservation status and guide the development of informed management strategies/policies.