The ship rat invasion of Big South Cape Island/Taukihepa in the 1960s was an ecological catastrophe that marked a turning point for the management of rodents on offshore islands of New Zealand. Despite the importance of this event in the conservation history of New Zealand, and subsequent major advances in rodent eradication and biosecurity, the source and pathway of the rat invasion of Big South Cape Island has never been identified. Using modern molecular methods on contemporary and historical tissue samples, we identify the mitochondrial DNA (mtDNA) haplotype of ship rats (Rattus rattus) on Big South Cape Island and compare it to that of ship rats in the neighbouring regions of Stewart Island/Rakiura and southern New Zealand, all hypothesised as possible source sites for the invasion. We identify two haplotype clusters, each comprising three closely related haplotypes; one cluster unique to Stewart Island, and the other found in southern New Zealand and elsewhere. By a process of elimination we rule that the ship rat invasion of Big South Cape Island was neither by swimming nor boat transport from Stewart Island, and is unlikely to have come from the south coast ports of New Zealand. However, because the ship rat haplotype found on Big South Cape Island is cosmopolitan to New Zealand’s South Island and elsewhere, we can only confirm that the invasion likely originated from some distance, but are not able to identify the invasion source more precisely. An unexpected consequence of our study is the discovery of five new mtDNA haplotypes for R. rattus that have not been previously reported