In this study, we examined the genetic diversity in three populations of the critically endangered annual, Bidens cernua, in Japan by using inter-simple sequence repeat markers and compared our data with those from two common congeners: Bidens radiata var. pinnatifida and Bidens tripartita. In contrast to our expectations, the degree of genetic diversity at the species level was higher in B. cernua than in B. radiata var. pinnatifida or B. tripartita. At the population level, the degree of genetic diversity was highest in B. cernua.These results may be ascribed to the mating system and method of seedbank formation in B. cernua. An analysis of molecular variance revealed relatively high genetic differentiation among the populations of all three species. We concluded that the distribution width could not be an index of genetic variability in Bidens examined in this study.