The genus Desmodesmus (Chodat) S.S. An, T. Friedl & E. Hegewald is ubiquitous in freshwater ecosystems, such as rivers, ponds, and wetlands. The actual species diversity and distribution of the genus is unknown because of morphological plasticity affected by habitats. Currently, 38 Desmodesmus species have been reported in Korea most of which transferred from the genus Scenedesmus recently, however, no phylogenetic relationships have been studied yet. Despite the challenges in analyzing relationships among Desmodesmus species through the morphology, ecology, and original description, this study focused on examining species-level relationships using the FBCC culture strains isolated from Korea. A total of 299 sequences (66 of 18S rRNA, 47 of atpB, 67 of petA, 52 of rbcL, and 67 of tufA) were newly determined and used for phylogenetic analysis. Four plastid genes tend to have higher variation than 18S rRNA in the variable sites and P-distance. From the combined phylogeny, the Desmodesmus included six clades such as Clade-1: D. pseudoserratus and D. serratus, Clade-2: D. communis, D. dispar, D. maximus, D. pannonicus, unidentified Desmodesmus sp., Clade-3: D. bicaudatus and D. intermedius, Clade-4: D. microspina, D. multivariablis, D. pleiomorphus, D. subspicatus, Clade-5: D. abundans, D. kissii, and D. spinosus, and Clade-6: D. armatus, D. armatus var. longispina, D. opoliensis, unidentified Desmodesmus spp. The new sequence data from FBCC strains will be used to identify species and study the molecular ecology of scenedesmacean green algae in freshwater ecosystems. The phylogenetic information from this study will expand our understanding of Desmodesmus species diversity in Korea.