Drug resistance has become an extremely serious problem worldwide. Antibiotic resistance genes (ARGs) entering the environment with wastewaters promote replenishment of the resistome of natural microbioms. Distribution of several clinically significant ARGs in wastewaters of Rostov-on-Don (Southern Russia), lower reaches of the Don River and natural waters of the neighboring region was investigated. Metagenomic DNA samples isolated from 250 ml of wastewaters or natural waters and 200 mg of surface sediments were used for the study. Identification of the ARGs was carried out with end-point detection PCR. Presence of NDM, OXA-48, CTX-M, VanA, VanB, ErmB, and TetM/TetO genes was detected in urban wastewaters. Samples of wastewater treatment plant (WWTP) sewage were enriched with ARGs in contrast to non-treated wastewaters from the sewage collector. NDM, VanA, ErmB, TetM/TetO genes were found only in wastewaters and were absent in samples of natural waters and surface sediments. Only OXA-48, VanB and CTXM genes were found in natural waters and surface sediments. The described ARGs are quite typical for urban and hospital wastewaters. The target ARGs were detected in the samples connected to the anthropogenous sources of pollution such as Rostov municipal WWTP or livestock enterprise effluents.