Populations of plants and animals at the edge of a species’ range may exhibit lower genetic diversity due to a combination of historical (e.g., postglacial dispersal) and contemporary (e.g., low effective population size, strong genetic drift or bottlenecks) processes. In this study, we analysed the genetic structure of populations of the European pond turtle (Emys orbicularis) in the northern part of the Pannonian Basin (Slovakia, Hungary, Austria), which represents the edge of the species’ range. Using mitochondrial DNA (mtDNA) and microsatellite markers, we aimed to assign individuals and populations into phylogeographic lineages and to determine genetic variation and population structure. Our study revealed that most individuals from natural populations belong to mtDNA lineage II (widely distributed haplotype IIa, and likely endemic haplotypes IIl and IIo), which is typical for the whole Pannonian region. However, non-native haplotypes were detected in one Slovak population (haplotype Ib) and in the Austrian Donau Auen National Park (haplotype IVa). Microsatellite markers revealed high variability comparable to that in other parts of the range, suggesting that marginal populations may not necessarily have reduced genetic diversity. A genetically mixed population was found in the Donau Auen National Park, consisting of turtles belonging to mtDNA lineage II (taxonomically assessed as subspecies E. orbicularis orbicularis) and lineage IV (taxonomically assessed as E. orbicularis hellenica), which is native to the circum-Adriatic region. The admixture suggests intense hybridization and introgression between the two lineages (subspecies).