In recent decades, the relationships between environmental conditions and community structures of stream macroinvertebrates have been investigated in many parts of the world. However, knowledge about assemblages of mayflies and other stream macroinvertebrates in Siberia (northern Asia) and Asia is limited. In fact, the patterns in mayfly species richness and assemblage structure in relation to environmental parameters have not been previously examined in western Siberia. The aim of the present study was to examine the relationship between Ephemeroptera community structure and physical parameters along a river altitude/longitude gradient in Siberia. The results showed that maximum species richness was at relatively low altitudes, high water temperatures, slow current velocities, medium stream widths, medium-small substrate particle size, and the presence of macrophytes. The mayfly assemblage was separated using TWINSPAN classification into eight distinct groups, which differed significantly with respect to at least one measured environmental factor. Multivariate ordination (detrended correspondence analysis) revealed that mayfly assemblages are structured by a single dominant gradient of altitude-related environmental variables; altitude and water temperature were the best predictors. Ordination further revealed that mayfly assemblages are structured by altitude-related environmental factors at high elevations, whereas in the lowlands these factors are less important.