Ascidians are marine filter-feeder chordates. Botrylloides ascidians possess diverse biological properties such as whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification difficult. Botrylloides diegensis is an ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii owing to the high morphological similarity between the sister species. More available sequences and strategies for identification would help resolve some of the confusion surrounding its ambiguous nature. We collected several Botrylloides samples from seven locations in Aotearoa New Zealand (Dunedin, Christchurch, Picton, Nelson, Whangateau, New Plymouth, and Invercargill) and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed the presence of three Botrylloides species: B. diegensis, Botrylloides jacksonianum, and Botrylloides affinis anceps.