Distinguishing between anthropogenic introductions and natural colonizations can be complex, especially in groups that are evidently both capable natural colonizers and anthropophilic, such as some geckos of the genus Hemidactylus. However, such information is fundamental both for constructing appropriate conservation approaches, as well as to identify biogeographical patterns. Here we assessed the origins and taxonomic status of the Hemidactylus geckos found on the Îles Éparses, disjunct small islands of the western Indian Ocean located around Madagascar, using partial mitochondrial DNA sequences. Hemidactylus platycephalus was confirmed on Juan de Nova, presumed introduced from continental Africa. The H. mabouia-mercatorius complex was identified on Juan de Nova, Europa, Tromelin, and Grande Glorieuse, with H. mabouia (sensu stricto) on Juan de Nova and H. mercatorius on Europa, from where it is likely to have been introduced to Tromelin. An unnamed lineage within H. mercatorius, previously known only from the Aldabra Islands, was identified on Grande Glorieuse, and further demonstrated the unexpectedly high genetic diversity within this lineage.