The population genetic structure of freshwater organisms depends upon the arrangement of streams, the extent and connectivity of ponds and marshes, as well as terrestrial dispersal capability. We assessed the population genetic structure and patterns of gene flow for a total of 324 European pond turtles Emys orbicularis sampled in ponds along the Strymon River, northern Greece. Analysis of mitochondrial DNA (mtDNA) control region sequences and nuclear DNA species-specific microsatellite loci recorded a high level of genetic diversity and suggested gene flow following a recent history of colonization. This pattern was advocated by an overall low-level nuclear DNA differentiation, limited but significant sequence divergence among mtDNA haplotypes, a significant level of genomic admixture across central ponds, and high proportions of individual turtle genomes from downstream and upstream ponds belonging to the same genetic cluster as defined by Bayesian analysis. Male-mediated gene flow emphasized the role of the Strymon River in turtle dispersal but did not rule out short-distance terrestrial movements. This work contributes to fine-scale population structure studies of freshwater organisms in habitats affected by human activities and provides information to help devise management and conservation plans for E. orbicularis in the Lake Kerkini National Park wetlands system.