Paedogamy (fusion of gametes produced within the same gametangium following meiosis) has rarely been reported in diatoms, with fewer than 10 confirmed examples. One of these, reported by L. Geitler, was in a diatom from Illmitz, Lake Neusiedl (Austria), identified as ‘Nitzschia frustulum var. perpusilla’. We observed uniparental auxosporulation in two Nitzschia clones isolated from the lower Ebro River (Catalonia, Spain), morphologically similar to Geitler's material and belonging to the N. inconspicua species complex. We established that the auxospores were formed paedogamously by Feulgen staining of the nuclei and time-lapse microscopy of living cells. However, reinvestigation of Geitler's original cytological preparations revealed differences between the Illmitz and Ebro material with respect to the length of the initial cells, the structure of the perizonium, and the timing of degeneration of superfluous haploid nuclei during gametogenesis, indicating a genetic and possibly a taxonomic separation. Scanning electron microscopic studies of Ebro auxospores revealed a novel form of longitudinal perizonium with bilateral asymmetry, and also scaly incunabula surrounding the unexpanded zygote, which contrast with the strip incunabula of another paedogamous Nitzschia species, N. fonticola. Molecular phylogenies, based on rbcL and partial LSU rDNA sequences, and evaluation of trees constrained to make the paedogamous species monophyletic, indicate that paedogamy probably evolved at least twice independently in Nitzschia sect. Lanceolatae, in the N. inconspicua and N. fonticola lineages.