Investigating sources of microbial contamination in a stream, the objectives of this study were to identify the sources of microbes in a river and quantify their relative contributions to the microbial community in the river. A microbial source tracking method relying on the 16S rRNA gene was used to investigate the microbial communities in water samples of a river. A total of 168 samples were collected from different pollution sources (livestock, domestic sewage treatment plants, industry, cropland, tributary and the sediment of the river), and 33 water samples were collected from eleven sites along the Chishui River in China. Our study revealed that in the river, the prevailing environmental factors across time and space structured and influenced the adaptation of bacterial communities to specific ecological niches.