Few reports of microbial groups associated with the groundwater flow system are available in China. 16S rRNA gene library was constructed by the cultured-independent approach to investigate gene sequences of microorganism in groundwater samples from the recharge (R), intermediate (M) and discharge (D) zones of an experimental watershed at Zhuhai campus of Sun Yat-sen University. Proteobacterium, Candidate division OPx, uncultured archaeon (uncultured Crenarchaeote and Euryarchaeote) and Actinobacterium are found predominant in all these three zones with the Proteobacterium accounting for 23.21%, 36.21%, and 28.84% in R, M, D zone respectively. The other predominant microbial groups were identified for varied zones, e. g. Eubacterium and Nitrospira in the R wells, Eubacterium and Acidobacterium in the M wells, and Bacteroidetes bacterium in the D wells. Linkages and potential evolution of microbial groups among three zones were examined by using the genetic neighbor-joining tree. Environmental adaptation along the groundwater flow contributes to the similarity and discrepancy of microorganism in term of the genetic tree, and the ecological functions of the microbial groups need further assessment.