AbstractSampling is often standardized with regard to time and space to monitor population characteristics. Effective sampling of large reservoirs can be complicated by the inherit bias of habitat variability along the reservoir gradient. Our objective was to assess spatial and seasonal distribution patterns of target and nontarget fish species in a large reservoir to identify potential sources of bias. A multitude of fish community and habitat characteristics were measured during seasonal (spring, summer, and fall) gill‐net surveys in 2015 and 2016 at Lake McConaughy, Nebraska. Spatial assessments along the longitudinal gradient (up‐reservoir to down‐reservoir) and between opposing shorelines were made for fish metrics from the seven most common species, and associations between species‐specific relative abundance and habitat data were examined. Spatial differences in relative abundance and size structure occurred for nearly all species, with the greatest spatial variability occurring between the upper and lower reservoir zones, but few strong correlations were observed between species‐specific relative abundances and habitat data. Seasonal differences occurred in relative abundance and size structure for most species, and size structure was lowest in the fall for most species. To reduce the impact of spatial variability, we determined the minimum required sampling effort to achieve precise estimates of mean catch per net‐night using two resampling strategies, with 16 or fewer gill‐net sets required for five of seven species and 14 or fewer required if a stratified random approach is utilized. This research quantifies the extent of spatial and temporal variability in standardized sampling on a large reservoir and provides novel insight for managers of similar systems considering a stratified random approach to reduce sampling effort while maintaining desired levels of precision.