Zygnema is a conjugating filamentous green algal genus that is distributed in a broad range of freshwater habitats, from sea level to alpine summits. Although more than 150 species have been described worldwide, their taxonomy remains unclear, probably owing to their relatively simple morphology. We investigated the detailed morphology of Korean Zygnema species, combined with analysis of the plastid psbA gene from 22 specimens of the genus and putative relatives, in order to develope a key to their identification and isolation, and to determine their relationships. We recognized two species of Zygnema; Z. insigne and Z. leiospermum, based on morphological characters such as width of the vegetative cell, position of zygospores, dimensions and form of spores, shape of female gametangia, and color of mesospores. The analysis of psbA data was consistent with morphological comparison. The pairwise divergence between two species was 3.7-4.1% (34-38 bp) in psbA sequences. The phylogeny of psbA revealed the monophyly of Z. insigne and Z. leiospermum together with two isolates of Z. circumcarinatum from Germany and Scotland. This is the first report on the psbA gene phylogeny of Zygnema.