Abstract: In the groundwater contamination research, molecular biotechnologies are one of the cutting-edge technologies. This kind of technologies have been commonly used at site scale, but rarely applied at regional scale. The shallow groundwater in Hutuo River Basin was selected as the study subject, because of it is one of the typical regions in front of the Taihang Mountains.67 samples were collected along the river. The 16s rDNA gene sequences were tested by high-throughput sequencing technology. According to the environmental factors of NO₃^-, COD and TDS, the microbial communities and the functional indicator microorganisms related to groundwater contamination were studied. The results showed that, using the cumulative probability distribution method, the samples were divided into three groups:background (B group), nitrate pollution (N group), and organic pollution (Y group).This grouping rule was close to the Ⅲ water grade threshold in the quality standard of groundwater. The order of microbial community richness is B group > N group > Y group. The organic pollution could decrease the richness diversity of microbial communities, and the difference was more significant in comparison with background microorganisms. The functional indicator microorganisms related to organic pollution was Acinetobacter, and the functional indicator microorganisms related to nitrate pollution was Nitrospira. The molecular biotechnologies and analysis methods used in this research could provide the theoretical evidence for regional environmental investigation and bioremediation.