The pond slider Trachemys scripta is one of the most widespread alien turtle species in the world. Its unregulated trade and the consequent uncontrolled releases into the wild led to negative impacts on the native turtles of the invaded areas. In Italy, alien pond sliders are widely spread, and the occurrence of hatchlings and well-established populations is known in some areas. However, to date in Sicily, only a single female of Trachemys scripta laying eggs was reported. Besides that, nothing is known about the actual reproduction success and establishment of self-sustaining T. scripta populations in Sicily. Therefore, based on 14 previously characterised highly polymorphic microsatellite loci, we aimed to unravel if the alien turtles successfully reproduce in Sicily, in both natural and semi-natural areas (i.e., lake “Biviere di Gela” and “Parco d’Orléans”, respectively). Our results show a clear structuring of the two studied populations. Several full-sibling relationships between the sampled turtles were found, suggesting actual reproduction in the wild of the pond sliders in Sicily. However, further sampling is desirable since no parent-offspring relationships were observed in the studied populations. Due to our results, systematic monitoring of alien pond sliders in the whole of Sicily is crucial for better planning of mitigation strategies in order to protect the native biota of Sicilian inland waters.