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Aim: To evaluate the ability of precipitation‐based environmental DNA (eDNA) sample collection and mitochondrial 12S metabarcoding sequencing to reconstruct well‐studied fish communities in lakes and rivers. Specific objectives were to 1) determine correlations between eDNA species detections and known community composition based on conventional field sampling, 2) compare efficiency of eDNA to detect fish biodiversity among systems with variable morphologies and trophic states, and 3) determine if species habitat preferences predict eDNA detection. Location: Upper Great Lakes Region, North America. Methods: Fish community composition was estimated for seven lakes and two Mississippi River navigation pools using sequence data from the mitochondrial 12S gene amplified from 10 to 50 water samples per waterbody collected in 50‐mL centrifuge tubes at a single time point. Environmental DNA (eDNA) was concentrated without filtration by centrifuging samples to reduce per‐sample handling time. Taxonomic detections from eDNA were compared to established community monitoring databases containing up to 40 years of sampling and a detailed habitat/substrate preference matrix to identify patterns of bias. Results: Mitochondrial 12S gene metabarcoding detected 15%–47% of the known species at each waterbody and 30%–76% of known genera. Non‐metric multidimensional scaling (NMDS) assessment of the community structure indicated that eDNA‐detected communities grouped in a similar pattern as known communities. Discriminant analysis of principal components indicated that there was a high degree of overlap in habitat/substrate preference of eDNA‐detected and eDNA‐undetected species suggesting limited habitat bias for eDNA sampling. Main conclusions: Large numbers of small volume samples sequenced at the mitochondrial 12S gene can describe the coarse community structure of freshwater systems. However, additional conventional sampling and environmental DNA sampling may be necessary for a complete diversity census. [ABSTRACT FROM AUTHOR]