The aim of this study was to develop and validate a high resolution melting (HRM) method for the rapid, accurate identification of the various harmful diatom Pseudo-nitzschia species in marine environments. Pseudo-nitzschia has a worldwide distribution and some species are toxic, producing the potent domoic acid toxin, which poses a threat to both human and animal health. Hence, it is important to identify toxic Pseudo-nitzschia species. A pair of primers targeting the LSU rDNA of the genus Pseudo-nitzschia was designed for the development of the assay and its specificity was validated using 22 control DNAs of the P. calliantha, P. delicatissima/P. arenysensis complex and P. pungens. The post-PCR HRM assay was applied to numerous unidentified Pseudo-nitzschia strains isolated from the northwestern Adriatic Sea (Mediterranean Sea), and it was able to detect and discriminate three distinct Pseudo-nitzschia taxa from unidentified samples. Moreover, the species-specific identification of Pseudo-nitzschia isolates by the HRM assay was consistent with phylogenetic analyses. The HRM assay was specific, robust and rapid when applied to high numbers of cultured samples in order to taxonomically identify Pseudo-nitzschia isolates recovered from environmental samples.