Monitoring reproductive migration is essential for the conservation of anadromous species. Shishamo smelt (Spirinchus lanceolatus) is endemic to Hokkaido, the northernmost large island in Japan. S. lanceolatus is an anadromous species that is known to migrate into rivers for a very short period in early winter. While this species has a special value for local fisheries, the catch amount has drastically declined in the last few decades. Information about S. lanceolatus reproductive migration dynamics is limited, which prevents them from being efficiently managed as a resource. In this study, we used environmental DNA (eDNA) methods as a noninvasive molecular tool for estimating presence/absence and abundance/biomass of S. lanceolatus during their migration into rivers. We developed a species-specific qPCR system for S. lanceolatus, examining (a) temporal variation in S. lanceolatus eDNA concentrations compared with catch data gathered by traditional methods and (b) variability of migratory patterns among river systems. In a core river for their spawning migration, we consistently detected S. lanceolatus eDNA throughout the spawning season, and the temporal distribution of eDNA concentration was consistent with that of the number of migrating S. lanceolatus estimated by catch survey data. In addition, we were able to detect S. lanceolatus eDNA even from rivers without any official record of their migration. Among rivers with eDNA detection, the relative eDNA concentrations varied, indicating that the population biomass differs largely among the river populations. Our study suggests that eDNA detection systems are useful for tracking reproductive migration of S. lanceolatus at fine spatio-temporal scales.