Backpack electrofishing is a common method used to compare total species richness and relative abundance of stream fishes across space and time. However, as with any sampling method, it is important to evaluate the sampling effort necessary to capture patterns of variation in fish assemblage structure across samples. Thus, we evaluated the efficacy of single-pass versus multiple-pass backpack electrofishing for minnows and darters in intermittent prairie streams. We found that in 14 of 19 three-pass electrofishing samples, we detected all species during the first pass. The samples where we missed species on the first pass were in pools with six to nine species, suggesting a single-pass sample worked best for pools with lower species richness. We also found that both the raw abundance (i.e., catch rates) and rank abundance of four common species based on the first pass is highly concordant with the second and third passes. Nevertheless, differences in capture efficiency varied by species and density. In particular, our ability to deplete a species from a stream pool was highly variable when fish densities were low, and for Phoxinus erythrogaster, it was variable across all densities. Overall, our data suggest single-pass electrofishing can be used to detect spatial and temporal trends in abundance and species richness given standardized effort, but may not be representative of absolute population densities.