Assessing genomic diversity and population structure of non-indigenous species is crucial to develop adequate management strategies. However, in species with scarce material for DNA extraction, applying genomic techniques can be a difficult task. Here we set a protocol for small DNA samples combining whole genome amplification (WGA) and genotyping-by-sequencing (GBS). This protocol was applied to the worldwide invasive colonial ascidian Didemnum vexillum using a single zooid per colony. WGA–GBS performance was tested using half zooids, providing empirical demonstration for genotyping reliability. We analysed 296 individuals from 12 localities worldwide including native and the main invaded areas. Polymorphic loci datasets generated by locality, area and globally, identified genetic differentiation at all levels. The two groups found in Japan, the native area, matched Cytochrome Oxidase I clades and were strongly differentiated at the genomic level suggesting reproductive isolation. Our genomic analyses confirmed that only one clade spread worldwide. We also detected some clones, always within the same locality. Genetic diversity was high in both the introduced and in the native area. Three independent colonisation events determined the global distribution of the species, although population pairwise comparisons within each introduced genetic cluster were significant. Human-mediated transportation seems to drive the distribution pattern of this species worldwide and regionally, as there is a lack of isolation by distance within introduced areas. Diverse and well differentiated populations point to a high expansion potential of this worrisome worldwide invader.