Abstract Background The seasonal variations in macroinvertebrate communities in tropical, temperate and subarctic regions have been observed and well documented to date, but similar studies conducted in subtropical rivers at the regional scale are relatively rare. In this paper, the macroinvertebrate communities from the main tributaries in the Three Gorges Reservoir Catchment (TGRC) were investigated as a function of the four seasons to explore the temporal variations in macroinvertebrate communities and further tests the temporal stability of certain metrics that are based on macroinvertebrates under a routine bioassessment framework. Results The taxa richness reached the highest point in spring, followed by winter, autumn and summer. The taxa Chironomidae, Heptageniidae, Corbiculidae and Baetidae dominated the communities across seasons. The temporal variations in communities were mainly reflected in the changes in taxa proportions between seasons. The percentages of the taxa Heptageniidae and Baetidae were the highest in autumn (normal discharge period) and lowest in summer (high discharge period). The abundance of macroinvertebrates was the lowest in summer, increased in autumn and winter, and then decreased in spring. Natural fluctuations of aquatic ecosystems (temporal effects) resulted in variations that were apparent in macroinvertebrate-based metrics, such as EPT%, Baetidae%, Caenidae%, Ephemerellidae% and Hydropsychidae%. Conclusions The results of our study demonstrated that the macroinvertebrate communities in the main tributaries of the TGRC varied as a function of seasons. This variation was fundamentally similar to the seasonal patterns in subarctic and temperate streams. Different hydro-morphological characteristics and water quality during the high discharge period (summer), low discharge period (winter) and normal discharge period (spring and autumn) strongly affected the distribution patterns of macroinvertebrate communities. Discharge variation among seasons resulted in seasonal fluctuations in the density of macroinvertebrates. In the TGRC, autumn was the important hatching period for mayflies (Ephemeroptera). The variations in metrics related to macroinvertebrates indicated that temporal effects should not be neglected under a biomonitoring framework in future studies.