Pseudo-nitzschia americana and the two closely related species P. brasiliana Lundholm, Hasle & G.A. Fryxell sp. nov. and P. linea Lundholm, Hasle & G.A. Fryxell sp. nov. are described, based on morphology. The phylogenetic positions of P. americana and P. brasiliana are inferred from partial nuclear-encoded large-subunit ribosomal DNA and their toxin production is assessed. The new species P. brasiliana and P. linea form stepped colonies, whereas P. americana is single celled. Pseudo-nitzschia americana, P. brasiliana and P. linea possess morphological features typical of Pseudo-nitzschia and Fragilariopsis, differentiating them from Nitzschia. All three species possess valve shapes similar to many Fragilariopsis species and they seem to constitute a morphological continuum between Pseudo-nitzschia and Fragilariopsis. Molecular phylogenetic analyses showed P. americana and P. brasiliana to be sister taxa within a strongly supported monophyletic clade comprising Pseudo-nitzschia and Fragilariopsis. On the basis of both the morphological and phylogenetic results, we conclude that P. americana can be assigned to Pseudo-nitzschia, following previous uncertainty as to whether it might belong to Nitzschia. Both P. americana and P. linea have been observed as epiphytes on other diatoms, which might indicate physiological differences from P. brasiliana, which has only been found free-living. In addition, P. americana and P. brasiliana differ with respect to valve width (transapical axis), the density of the transapical ribs (interstriae) and fibulae and the structure of the girdle bands (copulae). The results of the phylogenetic analyses and differences in distribution support these morphological differences. Pseudo-nitzschia linea differs with respect to valve shape, density of interstriae and structure of the girdle bands.