Although diatom taxa have been observed and described for many years using light and electron microscopy, several taxa have called for some clarifications and taxonomic reassessments. This is the case for the order Cymbellales D.G. Mann, which is widely represented in freshwater. The phylogenetic relationships among taxa belonging to this order are not always clear because their taxonomic status has been repeatedly revised. In this study, diatom cells were isolated from rivers in Italy, Luxembourg, Portugal and Spain. In total, 21 18S rDNA gene sequences, representing six genera of Cymbellales (Cymbella C. Agardh, Didymosphenia M. Schmidt, Encyonema Kutzing, Gomphoneis Cleve, Gomphonema Ehrenberg and Reimeria Kociolek & Stoermer) were determined. These sequences were analyzed along with other known GenBank diatom 18S rDNA gene sequences. The results indicate that the Cymbellaceae Greville and Gomphonemataceae Kutzing, especially the genus Gomphonema, are paraphyletic, and that the significance of some of the morphological characteristics traditionally used for classification purposes requires a reassessment. These results also demonstrate the importance of a polyphasic approach combining both morphological and molecular data in attempting to improve the taxonomy and classification system of diatoms.