The characteristic of species composition of ichthyofauna of the upper Don basin, including data on moderate and small rivers, is provided for the first time. The examined part of the basin embraces territories of Tula, Ryazan, Lipetsk, Tambov, and Voronezh oblasts. A total of 51 species—66.7% of the total number of species recorded in water courses of the entire Don basin—were recorded in the ichthyofauna of the region. The decrease in the number of species in the upper Don basin as compared to its other sections proceeds mainly at the expense of marine and euryhaline species, as well as due to anadromous acipenserids. In this part of the basin, a smaller number of invading species was also recorded. However, Amur sleeper Perccottus glenii that was not discovered so far in other parts of the basin has dispersed here sufficiently widely. The wide distribution of bearded stone loach Barbatula barbatula and minnow Phoxinus phoxinus can be possibly also considered a distinguishing feature of fish population of the upper reaches of the Don in relation to other sites.