Nitellopsis obtusa (Charales, Charophyceae) are widely distributed from Europe to Asia; however, this species has been recorded in only the five lakes in central Honshu in Japan. This species was thought to be extinct in Japan, but was rediscovered in limited areas of Lake Kawaguchi in central Honshu. More recently, we discovered more Japanese populations of N. obtusa in Lake Biwa in western Honshu, and it became clear that the species had a broader distribution in Japan than originally believed. In addition, although only male or female thalli have been collected at each lake, both male and female thalli were found from Lake Biwa. This is the first report of a potentially sexual population of N. obtusa in Japan. The DNA sequences of three chloroplast DNA markers, including both coding and non-coding regions, were identical in all specimens from Lake Kawaguchi and Lake Nojiri (Central Honshu), and differed from those of Lake Biwa and German specimens. Although Japanese and German specimens were genetically similar, Japanese specimens displayed considerable genetic diversity according to locality.