In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze-thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR-DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR-DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.