We analysed the molecular and morphological features of strains of Chattonella subsalsa isolated from the western Adriatic coast (Mediterranean Sea), with the aim of confirming their classification and elucidating their phylogenetic positions within the Raphidophyceae. We sequenced parts of the ribosomal operon, including the small subunit (SSU), the internal transcribed spacer region (ITS) and the large subunit (LSU) of the rDNA. Additionally, we analysed sequences of the chloroplast-encoded subunit psaA of Photosystem I (PSI) and rbcL, encoding the large subunit of the Rubisco gene. For three phylogenetic markers (LSU, ITS, rbcL), the sequences of the strains from the Adriatic Sea were identical and for two markers (SSU, psaA) only minor differences occurred. All strains were sister to, but well separated from, sequences from isolates in culture collections and from GenBank, thus far classified as belonging to C. subsalsa. Light and electron microscopy provided evidence for morphological differences between a strain of C. subsalsa (CCMP217) from the Gulf of Mexico and the isolates from the Adriatic Sea. Differences concerned the shape and arrangement of chloroplasts and the presence of mucocysts and other surface microstructures, which were only observed in isolates from the Adriatic Sea. This is the first evidence for two different taxa classified as C. subsalsa, which are clearly separated on the basis of several genetic markers and also show morphological differences. As compared with strains assigned to C. subsalsa from the NCMA (formerly CCMP) culture collection, the Adriatic strains more closely match the original species description. This would imply that strain CCMP217 and other genetically similar strains should be described under a new species name. Nevertheless, given the high morphological plasticity of Chattonella species, the definition of the true C. subsalsa must be decided based on detailed morphological and molecular analysis of more strains from other geographical areas.