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Marine diatoms of the genus Pseudo-nitzschia are widespread and occur in various environments. About half of the species described today have been shown to be toxic, producing the potent neurotoxin, domoic acid. The Pseudo-nitzschia species are a common component of the Adriatic phytoplankton community and are occasionally predominant. In the present study, the composition of the Pseudo-nitzschia species was studied in four distinct aquaculture areas from February 2022 to February 2023. These are the first results of a comprehensive study of Pseudo-nitzschia diversity combining morphological and molecular analyses. The integrated approach resulted in the confirmation of Pseudo-nitzschia brasiliana, Pseudo-nitzschia hasleana, Pseudo-nitzschia galaxiae, and Pseudo-nitzschia linea. The species, P. galaxiae, was the most frequently occurring, P. linea was found in all investigated areas, while P. hasleana and P. brasiliana were rare, and their distribution was limited mainly to Kaštela Bay. The findings of P. brasiliana and P. hasleana represent the first confirmation for the Adriatic Sea, while P. galaxiae and P. linea have not been previously reported along the eastern Adriatic coast.