<p>Urbanization has promoted the development of human society, but is often accompanied by environmental pollution. To investigate the extent to which differing levels of urbanization might affect antibiotic heterogeneity and distribution in the aquatic environment, we contrast the Yongjiang Estuary, an area of intense urbanization, with the Baixi Reservoir- whose hinterland is much less urbanized. We found that antibiotic residues in Baixi Reservoir and Yongjiang Estuary were significantly different due to urbanization gradient. 14 antibiotics were detected in Yongjiang Estuary with an average concentration of 0.74 ng/L, and the dominant classes being was macrolides. 13 antibiotics were detected in Baixi Reservoir with an average concentration of 0.12 ng/L, and the dominant class being tetracyclines. Total nitrogen, total suspended particulate matter and dissolved organic carbon (DOC) were found to have strong effect on the concentration of quinolones in Baixi Reservoir. However, salinity, ammonia nitrogen, DOC and chloride ion concentration were found to have strong effect on the concentration of quinolones in Yongjiang Estuary. DOC was the common factor affecting the concentration of antibiotics. This study provides data which reveal the distribution characteristics of antibiotics under different urbanization level and elucidates the risks posed by antibiotics resistance associated with rapid urbanization occurring in China and throughout the developing world.</p>