Nitzschia sublinearis Hustedt and N. pura Hustedt are common oligosaprobic freshwater diatom species that frequently occur in diatom inventories, thus being important in water quality studies. Both are considered as species with overlapping diagnostic criteria in several floras, which is typical of the whole genus Nitzschia. The type material of Hustedt of N. sublinearis and N. pura was examined using LM and EM in order to document the range of variation within the type populations and to compare it with populations occurring in different European rivers. Detailed observations allowed recognition of two new freshwater diatom species: N. alicae sp. nov., occurring in mesotrophic up to eutrophic conditions, and N. puriformis sp. nov., mostly occurring in oligotrophic habitats, both in rivers and streams at middle and high altitudes. The most reliable taxonomic features that separate both new species from the most similar taxa are the density of fibulae and striae, valve shape, and valve width as well as the shape of areolae. Morphological examination of different populations indicates that N. puriformis is relatively common in European rivers and has been overlooked to date and confounded with N. pura by several researchers. By contrast, N. alicae has, to date, been collected only in Slovakia and Northern Italy, but with a high frequency of occurrence and sometimes in high abundance at sites.