In this study, with small shallow lakes in Huaihe River Basin as object, the biodiversity and distribution characteristics of ammonia-oxidizing bacteria (AOB) were analyzed. Simultaneously, to explore the role of different sources of nitrogen pollutants on the abundance and distribution characteristics of AOB, nitrate sources were investigated via molecular biological means and bacterial denitrification stable isotope technology. The results showed that the diversity and abundance of AOB were the highest in Desheng Lake, while those in the northern part of the Dazong Lake were the lowest, demonstrating that the complex pollution sources and surrounding environment of Desheng Lake and the dense aquatic plants in the center of Dazong Lake led to the increase of the diversity and abundance of AOB. Phylogenetic analysis of amoA gene showed that the two most abundant genera in Desheng Lake, Dazong Lake and Jiulongkou were Nitrosomonas (92.3%) and Nitrospirillum (7.7%). Based on the distribution of AOB, nitrate nitrogen and AOB changed consistent, and nitrate nitrogen in overlying water was high at the point with high AOB abundance. Isotope traceability analysis showed that long-term seine farming led to a decrease of δ15N (0.57%) and the long-term over accumulation of decaying plants led to an increase of δ15N (1.0%). The research is helpful for the further study of the nitrogen cycle and management of shallow lakes.