The Magdalena River Turtle (Podocnemis lewyana) is a Colombian endemic species, endangered due to human exploitation and habitat destruction. To date, this species is poorly known ecologically and data on its genetic diversity are lacking. Here we report on the first genetic survey of the species across its distribution range. We obtained mitochondrial DNA sequences (488 bp) of the cytochrome b gene from 109 individuals. Samples belong to populations located at several different localities, grouped in five regions, along the four main river basins: Magdalena, Cauca, San Jorge, and Sinú drainages. We found two haplotypes, which differ in only one nucleotide substitution and which are represented with different frequencies in the five geographic regions. These results suggest that P. lewyana harbors little genetic variation and is a genetically uniform species, but more variable markers (i.e., microsatellites) should be used to unravel fine-scale phylogeographic structures in this species.