Environmental DNA (eDNA) has been established as a noninvasive and efficient approach to sample genetic material from aquatic environments. Although most commonly used to determine species presence and measure biodiversity, eDNA approaches also hold great potential to obtain population-level genetic information from water samples. In this study, we sequenced a panel of multiallelic microsatellite markers from filtered water and fish tissue samples to uncover patterns of intraspecific diversity in the freshwater Round Goby (Neogobius melanostomus) across their invaded range in the Laurentian Great Lakes region. Although we found that the concentration of nuclear eDNA is lower than mitochondrial eDNA, we nonetheless detected over two-thirds of all alleles identified from genotyped tissues in our eDNA samples, with the greatest recovery of common alleles in the population. Estimates of allele frequencies and genetic variability within and between populations were detected from eDNA in patterns that were consistent with individual tissue-based estimates of genetic diversity and differentiation. The strongest genetic differentiation in both eDNA and tissues exists in an isolation by distance pattern. Our study demonstrates the potential for eDNA-based approaches to characterize key population parameters required to effectively monitor, manage, or sustain aquatic species.