Defining species in the brown algal genus Colpomenia is a challenging endeavour because of their morphological similarity, overlapping phenotypic variation, the absence of conspicuous diagnostic characters, and often lack of reproductive structures crucial for their identification. Thus, the use of molecular tools has become widely used to study Colpomenia taxonomy and evolution. The following four Colpomenia species are described along the Australian coast: C. claytoniae M.Boo, K.M.Lee, G.Y.Cho & W.Nelson, C. ecuticulata M.J.Parsons, C. peregrina Sauvageau, and C. sinuosa (Mertens ex Roth) Derbès & Solier. The objective of this study was to assess the diversity of Colpomenia species in southern and south-eastern Australia by using DNA barcoding techniques and single-marker species delimitation methods. We generated 44 new COI-5P DNA sequences from nine different populations across three Australian states (South Australia, Victoria and Tasmania), and applied 13 variations of four species delimitation methods (ABDG, SPN, PTP, GMYC). Our results recognised three Colpomenia species in the region, namely, C. sinuosa, C. claytoniae, and C. peregrina. Colpomenia sinuosa is the most widely distributed species in Australia. Colpomenia peregrina and C. claytoniae presented high levels of intraspecific genetic divergence. We did not find C. ecuticulata, although it has been previously reported from nearby our sampling area.