Background/Question/Methods 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 samples may also hold great potential to obtain population genetic information from water samples. In this study, we sequenced a panel of multiallelic microsatellite markers from eDNA and tissue samples to uncover intraspecific diversity in the nuclear genome of the round goby (Neogobius melanostomus) at 15 locations across their invaded range. We tested the similarity between eDNA-based and individual genotype-based estimates of allele frequencies and demonstrate a novel approach to estimate genetic diversity and differentiation from eDNA samples. Such information is used to evaluate pathways of invasion spread and identify likely source populations for newly invaded inland lakes. Results/Conclusions We demonstrate the capacity for next-generation sequencing of eDNA samples to detect microsatellite allele frequencies and allelic variation among populations of an invasive species. eDNA approaches detected alleles at similar frequencies to genotyped tissues, with correlations of up to r = 0.89 between the relative frequencies of eDNA sequences and the relative frequencies of alleles in sampled individuals. Genetic variability within and between populations was also detected from eDNA in patterns that were consistent with individual tissue-based estimates of genetic diversity and differentiation. Our study demonstrates the potential for eDNA-based population genetics to characterize key population parameters required to effectively monitor, manage, and sustain aquatic species. Such non-invasive genetic sampling will have a strong role in addressing the need for rapid and cost-effective population-level genetic assessments for use in ecological and conservation research.