The karstic nature of the Yucatan Peninsula promotes the formation of submerged caves and sinkholes that are inhabited by an endemic subterranean water fauna. By contrast with most other micro-endemic stygobitic species, the freshwater palaemonid shrimp Creaseria morleyi is widely distributed across the northern part of the peninsula. In the present study, we investigated the phylogeographic structure of C. morleyi using two mitochondrial genes as markers, and explored hypotheses related to its evolution in the peninsula. DNA from 14 localities was extracted, and the 16S rRNA and cytochrome oxidase subunit I (COI) genes were amplified and sequenced. The different haplotypes were identified to construct a haplotype network and perform a nested clade analysis. Five haplotypes of the 16S gene were obtained, with a maximum divergence of 0.5%. One of these haplotypes is widely distributed and the most divergent is located in the north-western section of the peninsula. Twelve haplotypes for the COI gene were found with a maximum divergence of 2%, showing the same spatial pattern. The analysis revealed two significantly different clades corresponding to populations in the centre and south-east of the peninsula as a consequence of restricted genetic flow with isolation-by-distance. The divergence time of these two clades was 40–120 thousand years. The genetic variation in C. morleyi, the relationship between haplotypes and their geographic distribution, along with the geological history of the Yucatan Peninsula, may indicate that this variation is a relict of an ancient marked genetic structure reduced by changes in sea level that resulted in a series of bottlenecks. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 315–325.