Podocnemis lewyana is an endangered endemic river turtle of Colombia. Using ten unlinked polymorphic microsatellite loci and a 691-bp-long DNA fragment corresponding to the more variable portion of the mitochondrial control region, we investigated genetic diversity and population structure throughout its range. Both neutral markers showed extremely low diversity and weak population differentiation. Our data indicate that the genetic history of P. lewyana has been impacted by multiple bottlenecks and population expansion since the Pleistocene. The observed differentiation pattern is most likely the result of historically low genetic variation resulting from restricted geographic range and aggravated by recent anthropogenically induced bottlenecks. Based on slight differences in allele frequencies among populations, we suggest that three regions should be treated as demographically independent Management Units in order to preserve maximal genetic diversity: (1) the Upper Magdalena River Basin, (2) the Lower Magdalena + Lower Cauca + San Jorge River Basins, and (3) the Sinú River Basin. Among the Management Units, only low to moderate levels of gene flow were detected; these are largely unidirectional from Management Units 1 and 3 into Management Unit 2.