Fish assemblage in the Úpoř brook was observed. The stream was inhabited by 13 fish species and Lampetra planeri. The abundance reached the mean value of 12175 spec.ha-1 and the mean biomass 395 kg.ha-1. Salmo trutta dominated, Cottus gobio was also very abundant and inhabited preferably riffles as well as Barbatula barbatula. Leuciscus cephalus and L. leuciscus were resident in pools. Other species occurred occasionally. Preferences in habitat and microhabitat use were evaluated using RDA and FE test. Two first axes of RDA explain 27.8% of total variability of all species, and 95.3% of relationship between the species and the environmental variables. These factors significantly correlated with the axes: distance from the mouth, maximum depth, presence of mud, presence of stones as shelters, presence of rocky shelters, and presence of the deposits. The character of substrates was the most important factor influencing the distribution of S. trutta, L. cephalus, L. leuciscus and B. barbatula, whereas C. gobio were greatly influenced by character of shelters. Also the distance from the mouth play a role in distribution of two last species. Seasonal changes in habitat preference of C. gobio were recorded.