Natural populations of Zygnema were collected from 80 stream sites across California, and eight species were identified and characterized morphologically. Generic and infrageneric concepts of Zygnema and Zygogonium were tested with cox3 and rbcL gene sequence analysis. Strains of Zygnema were positioned in a single monophyletic clade sister to Zygogonium tunetanum Gauth.-Lièvre. In both the rbcL and cox3 phylogenies, strains of Zygnema formed two major clades. The first clade contained species that have zygospores with a blue-colored mesospore or akinetes with a colorless mesospore. The second clade contained species that have a yellow or brown mesospore. The existing taxonomic concepts for Zygnema classification are not consistent with our molecular phylogeny and do not correspond to natural groups. We propose that mesospore color may be useful in the infrageneric classification of Zygnema. Newly described Zygnema aplanosporum sp. nov. and Zygnema californicum sp. nov. have zygospores with a blue mesospore formed in the conjugation tube and separated by a cellulosic sporangial wall. Z. aplanosporum also possessed a combination of vegetative and reproductive features characteristic of Zygogonium, such as presence of short branches, rhizoidal outgrowths, thickened vegetative cell walls, purple-colored cell content, small compressed-globular chloroplasts as well as predominant asexual reproduction. Z. aplanosporum and Z. californicum were deeply embedded in a larger clade of Zygnema both in rbcL and cox3 analyses. Based on our observations, there are no features or combination of features that separate Zygnema and Zygogonium. Therefore, we conclude that Zygogonium is probably a synonym of Zygnema.