Genetic introgression has recently become an important concern for conservation genetics as it can occur between rare and related common species, between various genetic groups and between individuals from different origins. Our aims were: (i) to determine whether hybridisation or introgression occurs between Emys orbicularis subspecies in a natural contact zone in France; (ii) to determine the geographic extent of the contact zone between distinct subspecies in France; (iii) to evaluate at which level introgression occurs, and finally; (iv) to evaluate whether combining mitochondrial and nuclear genetic markers reflects the same contact zone as when only one genetic marker is used. Introgression was evaluated by genotyping biparentally inherited microsatellites and sequencing the maternally inherited cytochrome b gene of French populations. We demonstrated strong introgression between subspecies under natural conditions in the old contact zone in southern and eastern France. Our results corroborated that introgression reflects past natural events, but also demonstrated that human impact has altered these patterns. We finally confirmed that the combination of mitochondrial and nuclear genetic markers is more appropriate to reveal introgression than the use of only one genetic marker.