The taxonomic criterion of Antithamnion sparsum was reappraised in comparison with A. densum and A. defectum based on crossing experiments, morphological observation, chromosome study and Random Amplified Polymorphic DNA (RAPD) analysis. These species had a very similar morphology but were sexually isolated. The chromosome number was n = ca. 24 for A. densum, n = ca. 21 for A. defectum, and n = ca. 44 for A. sparsum. All isolates of A. sparsum and A. densum showed polysiphonia-type life history Asexual reproduction was induced by favorable environmental conditions. In A. sparsum, 1-2% of male plants developed mitotic tetrasporangia together with spermatangia. In A. densum, 5-10% of tetraspores developed into asexual tetrasporophytes. Phylogenetic relationships between these species were examined using RAPD analysis, and A. glanduliferum was used as an outgroup. A total of 167 polymorphic RAPD markers amplified from 15 different primers were analyzed. Results suggested that these species were closely related, with A. defectum placed in the middle of A. sparsum and A. densum. Chromosome study and RAPD analysis implied that A. sparsum first separated from A. defectum through polyploidization and later A. densum evolved. These species may present another example of the narrow species concept in the genus Antithamnion.