The freshwater green algal genus <i>Desmodesmus</i> contains many cryptic species that are difficult to identify using morpho-anatomy. This study analyzed 310 ITS2 sequences related to <i>Desmodesmus</i> 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 <i>Desmodesmus</i>. These signature sequences had 18-33 nucleotides in length. This tool could exactly identify 19 species and 24 strain groups of <i>Desmodesmus</i>. In addition, a signature sequence, AGA GGC TTA AAC TGG GAC C, was specific for almost 90% of <i>Desmodesmus</i> strains and could represent the genus <i>Desmodesmus</i>. A strain MLC1 isolated from freshwater in Da Nang, Vietnam, was identified as <i>Desmodesus pseudoserratus</i> 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<i> Desmodesmus</i>.