The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.