Human-mediated secondary contact of recently diverged taxa offers valuable opportunities for studying the evolutionary mechanisms involved in the establishment and maintenance of genetic boundaries between taxa. We used mitochondrial and microsatellite markers to examine a recently introduced population of the spur-thighed tortoise (Testudo graeca) of mixed origin in the Doñana National Park (SW Spain). The earliest records of tortoises in Doñana trace back to the 18th century, but several population reinforcements in the 20th century with animals from Morocco are well-documented. Consequently, different genetic lineages, which represent distinct subspecies, are thought to co-exist there. Our results confirmed the presence of distinct lineages by revealing that tortoises of the subspecies T. g. marokkensis were introduced into a local allochthonous T. g. graeca population. Unexpectedly, T. g. marokkensis haplotypes exclusively appeared in males, and admixture levels were statistically sex-biased toward males. The sex ratio of the population deviated from parity, with males being 2.36-fold more abundant than females. Our results indicated that population reinforcements had a strong effect on the genetic composition of this population and aggravated its sex ratio deviation. We predict that this sex-biased pattern of introgression is ephemeral and advocated to the near loss of T. g. marokkensis haplotypes.