The genus Pleurastrum is a coccoid green alga comprising 10 species worldwide. Pleurastrum exhibits simple morphology and high polymorphism, which complicates the understanding of its diversity. We examined the morphological and ultrastructural characteristics of Pleurastrum using light, confocal, and transmission electron microscopy. Additionally, we performed phylogenetic analysis based on multigene sequences (nuclear SSU rDNA, 5.8S, internal transcribed spacer (ITS2) region, and plastid rbcL and tufA genes) from Pleurastrum strains to report two previously unrecorded freshwater species (Pleurastrum insigne and Pleurastrum microstigmatum) in Korea. The vegetative cells were predominantly spherical, with a few being ellipsoidal, and each cell contained a chloroplast with one pyrenoid. The sporangia produced several daughter cells, while the biflagellate zoospores were ellipsoidal and motile. Phylogenetic analysis confirmed that P. insigne and P. microstigmatum form well-supported monophyletic clades. Analysis of ITS2 secondary structures revealed similar patterns, with several differences in nucleotide sequences and insertions between the two species. The findings of this study expand the known distribution of Pleurastrum and enhance our understanding of its species diversity in Korea.