Ascidiella aspersa, originally native to the northeastern Atlantic, has emerged as a prolific invasive species in coastal waters worldwide. In 2010, it was identified as an alien species in Republic of Korea, rapidly colonizing artificial harbor structures and outcompeting native species. This study employs morphological analyses and genetic sequencing, focusing on mitochondrial DNA (cytochrome oxidase subunit I; mt-COI) and nuclear markers (18S rRNA), to unravel the genetic structure and haplotype diversity (Hd) of A. aspersa populations in Republic of Korea and globally. The analysis of 154 mt-COI and 127 18S rDNA global population sequences, as well as 80 mt-COI and 79 18S-rDNA Korean population sequences, revealed distinct genetic patterns. Among global populations, the mt-COI gene displayed significant genetic diversity, with 21 distinct haplotypes distributed across 41 polymorphic sites, which is indicative of extensive genetic variability. In contrast, the 18S rDNA marker exhibited limited diversity, with only four haplotypes identified at three polymorphic sites. In Korean populations, the mt-COI gene also exhibited substantial genetic diversity, with 14 distinct haplotypes displaying genetic variations at 29 polymorphic sites. Conversely, the 18S rDNA marker in Korean populations revealed a unique genetic pattern, with only one shared haplotype. These findings emphasize the complex genetic diversity within A. aspersa populations, both globally and in Republic of Korea. This genetic analysis provides valuable insights into the species’ colonization history and adaptation mechanisms, shedding light on the factors shaping its genetic structure. Further research is warranted to elucidate the ecological implications of these genetic patterns in the context of invasion biology.