•Existence of alternative microbiota states in rivers were identified by field data.•NH4+−N and NO3−−N were identified as main environmental stress.•Clostridiales, Nitrospirales and Myxococcales were discerned as key taxa.•Alternative stable states theory can guide river biodiversity conservation. Catastrophic shifts in river ecosystems can abruptly degrade their structures and functions, often reducing the efficacy of traditional remediation targeting physicochemical properties. Alternative stable states theory can not only explain this phenomenon but also provide a new insight into river restoration; however, little is known about the existence and implications of alternative stable states in a river. Considering the important role of benthic microbiota in sustaining river ecosystem structures and functions, ecological theory and high-throughput sequencing were combined to firstly investigate multi-stability in microbial communities and its relationship with environmental factors in river sediments. The Nanjing reach of the Yangtze River was selected as the study area because of its huge spatial heterogeneity and varying degrees of pollution. Bimodal distributions combined with temporal variations of microbiota status provided direct evidence of bistability by showing the instability at the intermediate. In addition, environmental stress, particularly concentrations of NH4+−N and NO3−−N, was identified as an important driver of alternative microbiota states from the perspectives of the behavior of bistable ecosystems. Comparison of α-diversity indices and network properties between two alternative microbiota states revealed that the diversity and co-occurrence pattern of microbial communities will be high if they are settled in favorable environments (i.e., comprehensive sediment quality identification index > 3.7). Key taxa, including Clostridiales, Nitrospirales and Myxococcales, were discerned by combining LEfSe and network analysis, and their strong interspecies interactions were believed to be an important factor in triggering alternative microbiota states. This study suggests alternative stable states theory should be considered in river remediation to better understand the response of river ecosystems to environmental stress and the effect of hysteresis, benefiting the implementation of effective monitoring and restoration strategies in a river of urban area. Display omitted