Culture strains of the Closterium moniliferum-ehrenbergii species complex were classified on the basis of three types of dormant spores: zygospores formed by homothallic and heterothallic conjugation and parthenospores formed without conjugation. Two parthenosporic strains of C. moniliferum from Hiroshima, Japan, and Ho Chi Minh, Vietnam, and two parthenosporic strains of C. ehrenbergii from Okinawa, Japan, and Ho Chi Minh, Vietnam, were used in the present study. Morphological observations on the process of parthenospore formation and germination were made using light and fluorescence microscopy. Parthenosporic strains of the two species essentially shared the same manner of parthenospore formation. Germination of parthenospores in C. moniliferum produced one germling without any meiotic event. The phylogenetic relationship between parthenosporic strains of the two species and among homothallic, heterothallic and parthenosporic strains were analysed on the basis of joint sequences of the 1506 group I intron and internal transcribed spacer-2 (ITS-2) regions of nuclear ribosomal DNA, using maximum likelihood. Phylogenetic analysis showed that parthenosporic strains of C. moniliferum and C. ehrenbergii were not monophyletic. Parthenosporic strains of C. ehrenbergii were in the same clade with heterothallic mating group K of the same species, whereas parthenosporic strains of C. moniliferum were sister to heterothallic mating group M of C. ehrenbergii. Inferred secondary-structure models of ITS-2 showed some common features among the three reproductive systems. The present study confirmed the hypothesis that parthenosporic populations have been derived from heterothallic populations in the C. moniliferum-ehrenbergii complex.