We tested for diel substrate use in artificial stream experiments for six minnows: redfin shiner (Lythrurus umbratilis), silver shiner (Notropis photogenis), spotfin shiner (Cyprinella spiloptera), central stoneroller (Campostoma anomalum), striped shiner (Luxilus chrysocephalus), and bluntnose minnow (Pimephales notatus). Our experimental pools contained two of three possible substrate types (sand, cobble, and gravel) per trial, and we tested if habitat use differed in day and night observations. Over 85% of individuals and all species were observed over cobble substrate during the day, and no species occurred in higher abundances over a single substrate type at night. Fish locations during night observations were over cobble and sand or cobble and gravel. Our results suggest that minnows use coarse substrates during the day and do not have substrate preferences at night. Habitat use studies with only daytime observations of small-bodied fishes likely cannot be interpreted as complete. Further investigations of substrate use variation with 24 h observations and additional body size or life stage variation will provide species-specific habitat requirements and better understanding of stream assemblage structure.