The European pond turtle (Emys orbicularis) is an endangered Old World species. The phylogeographic history of E. orbicularis has been extensively studied throughout its range. While local genetic variation holds valuable information for conservation efforts, specific regional details have yet to be thoroughly examined everywhere. The Valencian region on the Iberian Peninsula is one such area. Here, different evolutionary lineages of E. orbicularis meet, forming a part of the western Mediterranean hybrid zone. In this study, we comprehensively sampled all Valencian localities where E. orbicularis occurs. Using mitochondrial DNA and nuclear microsatellites, we determined the genetic structure of E. orbicularis populations. Our data reveals that the mitochondrial haplotype originating from the glacial refugium in the Apennine Peninsula is primarily restricted to the northern part of the Valencian region. Additionally, human-mediated dispersal processes may have contributed to the complex relationships between the evolutionary lineages of E. orbicularis in the natural hybrid zone. On a finer scale, E. orbicularis in the Valencian region can be classified into five distinct genetic populations. Morphometric analyses revealed sexual dimorphism in the plastron shape and slight variation among genetic populations. Notably, female, but not male, plastron shape correlates with individual heterozygosity. Given the ongoing threat posed by exotic turtles, we propose implementing targeted management strategies to mitigate the presence of alien turtles. These strategies should be directed towards localities that represent each genetic population within the region, reflecting the extensive population structure observed in our study.