The spread of non-indigenous species into new marine habitats represents an increasing threat to global diversity. Genetic techniques provide basic understanding of the invasion processes. The ascidian Microcosmus squamiger is considered to be native to Australia, having been spread worldwide via transoceanic vessels. It has successfully invaded artificial and natural habitats where it has become a pest. We studied phylogeography and genetic structure of 12 M. squamiger populations, including samples from its native range (Australia) and introduced populations from the Indian, Pacific, and Atlantic oceans, as well as the Mediterranean Sea. We amplified 574 bp of the mitochondrial COI gene in 258 individuals and found a total of 52 haplotypes. A haplotype tree revealed two main groups of haplotypes. The relative frequency of each group of haplotypes, multidimensional scaling, and analysis of molecular variance showed important differences between the western Australia localities and the remaining ones (eastern Australia and introduced populations). Furthermore, we found that the colonization of the different areas by M. squamiger has not occurred independently, as many introduced populations shared some low frequency alleles. A nested clade analysis showed a global pattern of restricted gene flow with isolation by distance, although we found episodes of long-distance dispersal in some clades. A contiguous range expansion was detected between Australian populations. We conclude that M. squamiger is native to Australia and has most likely expanded its range of distribution sequentially through worldwide shipping, especially from the harbours of the more populated eastern Australia. In introduced populations, we found a high genetic diversity which suggests enhanced invasive potential. Consequently, there is a need to control this species, as it outcompetes local biota and is an economic threat.