Periphytic algae exist widely in different waters. However, little is known about periphytic algae in long-distance water diversion channels across watersheds. We investigated the periphytic algae and the environmental factors at twenty sampling sites in the middle route of the South-to-North Water Diversion Project (MRP). The dominant species were Desmodesmus intermedius (Hegewald), Calothrix thermalis (Bornet & Flahault), Calothrix parietina (Bornet & Flahault) and Leptolyngbya benthonica (Anagnostidis) (dominance > 0.02) as measured in a whole year. Habitat heterogeneity in the MRP led to lower spatial heterogeneity and higher temporal heterogeneity of the periphytic algal community. Stochastic processes are the major process in periphytic community assembly. In deterministic processes, homogeneous selection had the major role in structuring the periphytic community, whereas the role of heterogeneous selection was less significant. In stochastic processes, dispersal limitations had the major role in structuring the periphytic community, whereas the role of homogenizing dispersal and drift were less significant. The variation in total nitrogen and total phosphorus promoted more stochastic processes (-1.96 < beta NTI < 1.96). The variations in water temperature and water velocity promoted more heterogeneous selection (beta NTI > 1.96). In integrating all of this empirical evidence, we explore the role of environmental factors in the action of ecological processes shaping thecommunity assembly of the periphytic algal community.