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Members of the genus Klebsormidium have cosmopolitan distribution and occur in a very wide range of freshwater and terrestrial habitats. Due to its simple filamentous morphology, this genus represents a taxonomically and systematically complex taxon in which phylogenetic relationships are still poorly understood. The phylogeny of Klebsormidium and closely related taxa was investigated using new ITS rRNA and rbcL sequences generated from 75 strains (isolated from field samples or obtained from culture collections). These sequences were analyzed both as single-marker datasets and in a concatenated dataset. Seven main superclades were observed in the analyses, which included sixteen well-supported clades. Some species of Klebsormidium, including the type species Klebsormidium flaccidum, were polyphyletic. Interfilum was recovered with high statistical support as sister taxon to a clade of Klebsormidium formed mainly by strains identified as K. flaccidum. Whereas some clades could be easily associated with described species, this was not possible for other clades. A new lineage of KIebsormidium, isolated from arid soils in southern Africa and comprising undescribed species, was discovered. Several morphological characters traditionally used for taxonomic purposes were found to have no phylogenetic significance and in some cases showed intra-clade variation. The capacity to form packet-like aggregates (typical of Interfilum), features of the morphology of the chloroplast and the type of habitat were the main phylogenetically relevant characters. Overall, Klebsormidium and Interfilum formed a more diverse algal group than was previously appreciated, with some lineages apparently undergoing active evolutionary radiation; in these lineages the genetic variation observed did not match the morphological and ecological diversity. (C) 2010 Elsevier Inc. All rights reserved.