The distribution of benthic macroinvertebrates can be studied at different scales. At a mesohabitat scale the variation in a benthic community structure is influenced by four major processes: 1) physical factors, 2) patchy food supplies, 3) reproductive and age related effects, 4) biological interactions. Even in a small part of a stream we find habitats with different environmental conditions and therefore the distribution of organisms is not uniform but reflects the characteristics of the habitats. We studied spatial distribution of benthic macroinvertebrates within a 100m long reach of a Strahler's first order stream Hadůvka (Czech-Moravian Highlands, Czech Republic). Several mesohabitats were distinguished there: fine sediment, pure sandy bottom, sand covered with leaves, gravel bottom, gravel covered with leaves and cumulated woody debris - debris dams. From each mesohabitat all substratum was taken using Hess sampler (area 0.03 m2, 100mm mesh size) into the depth 5cm. In a laboratory organic and inorganic matter was separated using decantation and elutriation. Mineral substrate roughness and the amount of organic matter were evaluated. The importance of substrate characteristics to the macroinvertebrate community structure was evaluated using multivariate analysis and species preferences for particular substrate were assessed.