© 2014, Springer International Publishing Switzerland. We investigate the genetic diversity of the sweeper Pempheris, a biological invader that entered the Mediterranean Sea via the Suez Canal. Two mitochondrial regions and one nuclear region were sequenced and topological reconstructions investigated from samples collected from the eastern Mediterranean Sea, Red Sea and three Indo-Pacific localities. Morphological and molecular analyses assigned samples from this study to three distinct species of Pempheris in the Red Sea (P. flavicyla, P. rhomboidea, and P. tominagai) and confirmed a misidentification of the Mediterranean sweepers, previously identified as P. vanicolensis and now recognized as P.rhomboidea. Pempheris rhomboidea clustered in a single clade including specimens from Madagascar and South Africa. Similarly to most other studied Lessepsian bioinvaders, no evidence of a genetic bottleneck in its invasive Mediterranean population was found. Yet, lowered gene flow levels were observed between Red Sea and Mediterranean populations in this species. These findings highlight the importance of molecular tools to the proper identification of morphologically challenging alien organisms and contribute to the understanding of the dynamics of Lessepsian invasions.