Although the number of morphological characters provided by diatom frustules for species identification is unparalleled among unicellular eukaryotes, morphology alone has in several cases proven to be insufficient to detect species boundaries in this algal group. Our recent nuclear rDNA sequence survey indicated that Cyclotella meneghiniana, one of the most intensively studied freshwater diatoms, is another example of a species complex. We therefore sampled genetic variation in the D1/D2 regions of the nuclear 28S rDNA and in a segment of the plastid encoded psaA gene in a larger sample of C. meneghiniana strains to assess genetic diversity and genetic structure over a wider geographic range within this diatom. A strict congruence of nuclear rDNA and psaA variants was observed, indicative of the presence of eight genetically distinct, cryptic lineages, four of which were not represented in our previous study. To test whether the genetic structure is caused by the complete lack of allogamy, or by the presence of reproductive barriers between allogamous cryptic species, we generated AFLP fingerprints from two subsamples of sympatric strains. Phylogenetic incompatibility in AFLP banding patterns did not differ significantly from panmictic expectations. Therefore, we suggest that C. meneghiniana is a complex of several, reproductively isolated, sexual species, rather than a complex of either strictly self-fertilizing or clonal lineages. © 2007 British Phycological Society.