Nemalionopsis tortuosa Yoneda et Yagi is a freshwater red macroalga recorded as a threatened species in Japan. Based on variations of branch length and branching density, 43 samples from nine Japanese populations of N. tortuosa were divided into two groups: one possesses short-branches and are densely branched (two localities), and the other one possesses short to long-branches and are sparsely branched (other localities). The variations of thallus and medulla diameters overlapped among the nine populations. Twenty six samples of N. tortuosa from 17 localities, including samples used in the morphological observations, had only 0-4 bp differences (0-0.35％ divergence) in the plastid encoded rbcL sequences. Four samples of the sparsely branched group and all five samples of the densely branched group had identical sequence (seq-A). The statistical parsimony network tree showed the haplotype seq-A as an early divergent lineage in this species. The two groups from the morphological analysis could not be recognized in the phylogenetic analysis, and we concluded that the two morphological groups belong to one taxon, N. tortuosa. Nemalionopsis tortuosa is one of the most rapid extinction of its population, and emergency protective actions are necessary for this species.