Since the first eDNA study reported in 2008, eDNA techniques have been developed as a non-invasive ecological tool for estimating presence/absence of living organisms in the wild. Yet, there are few studies reported on eDNA-based population genetics. In this study, we evaluated the applicability of an eDNA-based haplotype analysis on nagaremon-charr (an isolated morphotype of Salvelinus leucomaenis japonicus) in Anegawa river system that is known as one of the southern-most populations of charr species. For estimating haplotype compositions, we made a new primer set and applied it to semi-quantitative metabarcoding of eDNA samples. Our results indicate that populations of nagaremon-charr are composed of two haplotypes in the system, which are distinct from haplotypes of the normal morphotypes in the downstream population. The results of eDNA-based haplotype analysis were largely consistent with genetic structures that were reported in the previous study based on catch surveys. Furthermore, DNA concentrations estimated by semi-quantitative metabarcoding had a significant positive correlation to the estimated number of individuals (>1⁺) by mark-recapture surveys. These results suggest that the eDNA-based population genetics is indeed a powerful tool for understanding the current population status of living organisms in the wild.