| Summary: | <i>Botrylloides</i> species are important members of the fouling community colonizing artificial substrates in harbors and marinas. During monitoring in 2017–2020 of non-indigenous species in Korea, one colonial ascidian species was distinctly different from other native colonial ascidians, such as <i>B. violaceus</i> and <i>Botryllus schlosseri</i>, in South Korea. This species was identified as <i>B. diegensis</i>. DNA barcodes with mitochondrial COI were used to identify one-toned and two-toned colonies of <i>B. diegensis</i>. Intraspecific variations between Korean and other regions of <i>B. diegensis</i> from the NCBI ranged from 0.0% to 1.3%. The Korean <i>B. diegensis</i> was clearly distinct from other species of <i>Botrylloides</i> at 15.8–24.2%. In phylogenetic analysis results, Korean <i>B. diegensis</i> was established as a single clade with other regions of <i>B. diegensis</i> and was clearly distinct from Korean <i>B. violaceus</i>. After reviewing previous monitoring data, it was found that two-toned <i>B. diegensis</i> was already found in six harbors by July 2017. It has now spread into 14 harbors along the coastal line of South Korea. This means that <i>B. diegensis</i> might have been introduced to South Korea between 1999 and 2016.
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