Trinomys eliasi is an endangered species of spiny rat endemic to a small area in the lowlands of Rio de Janeiro state (Brazil). Limited data on its biology and variation are available for designing conservation policies. Here, we provide data on genetic variation of T. eliasi, elucidating aspects of its evolutionary differentiation based on analysis of cytochrome b DNA. Our findings showed that T. eliasi diverged from its sister species T. paratus in the early Pleistocene or late Pliocene (∼2.5 million years ago [mya]). Two T. eliasi mitochondrial lineages diverged in the early Pleistocene (∼2.1 mya) and are currently separated by a large river and Holocene areas previously occupied by a vast Pleistocene internal sea, which may have operated as a historical barrier between populations. The haplotypes of the southern lineage diverged relatively late in the Pleistocene (∼0.6 mya), and diversity of this lineage is not equally distributed across the landscape, but rather, it appears to be concentrated in the rainiest areas, which contain major forest remnants. This region should be considered a priority for conservation. In the northern extreme of Rio de Janeiro state, we found an isolated, highly divergent T. eliasi lineage, which deserves further investigation. This work highlights the need for fine-scale studies of genetic variation in endangered species for the preservation of their evolutionary diversity.