The headwaters of the Volga River exhibit large reaches with near‐pristine conditions, and therefore long‐term biodiversity monitoring of this catchment can provide rare and valuable information on a European lowland river. More specifically, freshwater fish species assemblages are a good indicator of ecosystem status, as they are particularly sensitive to environmental changes and hydromorphological alterations. Historical records show that the fish fauna of the Upper Volga has changed over time, both in species composition and in abundance. The construction of the Volga–Kama cascade (a series of large dams) has specifically affected the migration of diadromous species. Environmental DNA metabarcoding offers a non‐invasive approach to determine the number of species in an aquatic ecosystem, as well as their identity and distribution. This approach is especially useful for fish fauna surveys along large rivers and long‐term biomonitoring, with the advantage of having no impact on the species and their habitats. To infer the current fish species diversity and the spatial distribution of each species in the free‐flowing section of the Upper Volga River, as well as in selected tributaries, an environmental DNA metabarcoding approach was applied, using three mitochondrial DNA markers. This method allowed the positive identification of 23 fish species and their respective distributions in the headwaters of the Volga. This assessment provides a valuable example of the application of environmental DNA metabarcoding in a large river system, and constitutes a starting point for future investigations and long‐term biomonitoring in the Upper Volga system. In addition, the results can also serve as a reference for fish diversity assessments of other large European lowland rivers, and can guide future conservation and management measures in the headwaters of the Volga.