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Paracaprella pusilla Mayer, 1890 is a tropical caprellid species recently introduced to the Eastern Atlantic coast of the Iberian Peninsula and the Mediterranean Sea. In this study, we used direct sequencing of mitochondrial (COI and 16S) and nuclear (28S and ITS) genes to compare genetic differences in presumed native and introduced populations in order to infer its introduction pattern and to shed light on the native range of this species. The temporal pattern of genetic diversity at the westernmost limit of the geographic range of P. pusilla in Europe (the Atlantic coast of southern Spain) over an eight-year period was also investigated. Our results confirm P. pusilla as a neocosmopolitan species and suggest that the species is native to the Atlantic coast of Central and South America. Paracaprella pusilla seems to have been introduced into European waters from multiple introduction pathways and source populations, which are likely to include populations from coastal waters of Brazil. Multiple introduction pathways may have been involved, with the most important being commercial shipping through the Strait of Gibraltar. While this tropical species appears to be expanding in the Mediterranean, populations from the westernmost limit of its geographic range in Europe showed a temporal instability. This study constitutes the first molecular approach focused on this species, but it is also the first study of temporal change in genetic diversity of any introduced marine amphipod. Additional intensive sampling of this species, including both native and non-native populations, and detailed temporal studies are still necessary to properly understand how genetic diversity influences the introduction and survival of P. pusilla in invaded areas.