The freshwater green algal genus Desmodesmus contains many cryptic species that are difficult to identify using morpho-anatomy. This study analyzed 310 ITS2 sequences related to Desmodesmus species from GenBank and developed a new tool named DNA signaturing. DNA signaturing is a species identification method using signature sequences specific for each species or strain group within the genus Desmodesmus. These signature sequences had 18-33 nucleotides in length. This tool could exactly identify 19 species and 24 strain groups of Desmodesmus. In addition, a signature sequence, AGA GGC TTA AAC TGG GAC C, was specific for almost 90% of Desmodesmus strains and could represent the genus Desmodesmus. A strain MLC1 isolated from freshwater in Da Nang, Vietnam, was identified as Desmodesus pseudoserratus by this tool. DNA signaturing tool described here showed the highest efficacy for identifying strains and a remarkable ability to accurately classify cryptic species in the genus Desmodesmus.