Based on morphological variation found in specimens ascribed to Pseudo-nitzschia pseudodelicatissima and uncertainty regarding the delineation of P. pseudodelicatissima and P. cuspidata, cultures and field material of diatoms in the P. pseudodelicatissima/cuspidata complex were studied in morphological detail. Four different species were identified. The descriptions of the species P. pseudodelicatissima and P. cuspidata were emended on the basis of studies of type material. In addition, P. calliantha sp. nov. and P. caciantha sp. nov. were described as new species based on morphological and molecular data. The morphological differences among the species were found in characters such as width and shape of the valve, density of fibulae and striae, structural pattern of the poroid hymen, and structure of the girdle bands. The morphological studies were supported by phylogenetic analyses of the nuclear-encoded internal transcribed spacer 1, 5.8S, and internal transcribed spacer 2 rDNA of 24 strains representing 16 different Pseudo-nitzschia species. The description of the four species helps to explain the variation observed in mating experiments on cultures originally designated as P. pseudodelicatissima. At least two previous reports of toxin production in species identified as P. pseudodelicatissima have been identified as being caused by P. calliantha, and one additional report of toxin production has been identified as either P. pseudodelicatissima or P. cuspidata.