To determine the origin of introduced European populations of pumpkinseed (Lepomis gibbosus) – a freshwater sunfish, native to eastern North America that has spread across western and central Europe – we investigated the phylogeography of the species by sequencing the mitochondrial NADH subunit 1 gene. A total of 543 pumpkinseed were sampled from 32 sites across the native range of the species, and from 19 sites covering much of its introduced European range. The presence and geographic distribution of two distinct clades in North American populations were discovered, suggesting that pumpkinseed survived the Pleistocene glaciations in two refugia (Atlantic and Mississippian). Analyses of mitochondrial variation revealed that European pumpkinseed may have been introduced to Europe from at least two distinct sources, but from the Atlantic drainages of North America only, which is consistent with historical records. Within Europe, haplotype distributions of pumpkinseed lend support to the entry of non-native fishes into Iberia via the so-called ‘Perpignan–Barcelona corridor’. European populations possessed lower genetic diversity than populations from North America, suggesting that low genetic diversity may not preclude a species from establishing and spreading into new environments outside of their native range.