Proliferations of the genus Pseudo-nitzschia recur along the Catalan coast (NW Mediterranean) throughout the year. The establishment of 58 clonal cultures facilitated morphological studies with scanning electron microscopy (SEM) and ITS 5.8S rDNA sequence characterization. Moreover, strains of each species were examined with respect to sexual compatibility and toxicity. The results of the morphological and phylogenetic studies confirmed nine species of the genus Pseudo-nitzschia: P. brasiliana, P. calliantha, P. delicatissima clade A/del 2, P. arenysensis, P. fraudulenta, P. galaxiae, P. linea, P. multistriata and P. pungens clade I. Moreover, two Pseudo-nitzschia species, P. caciantha and P. cf mannii, could only be identified following SEM analysis of their morphology. None of the cultured strains of Pseudo-nitzschia analyzed produced domoic acid in amounts above the limit of detection. The current distributions of the Pseudo-nitzschia species in the Mediterranean Sea were plotted on maps, which led to the following observations: i) diversity within this genus is much greater than previously considered, ii) some species have a broad distribution (e.g. P. calliantha), iii) whereas the distribution of others is narrowly restricted (e.g. P. pungens clade I). Moreover, this study reports the first detection of P. linea in the Mediterranean Sea and is the first description of P. galaxiae and P. cf mannii along the Catalan coast. Morphological studies coupled with molecular biological characterization, mating tests and biogeographic distribution analyses provide a critical theoretical basis for testing and/or implementing the current phylogenetic framework in the genus Pseudo-nitzschia.