Infections caused by antibiotic-resistant bacteria (ARB) result in an estimated 1.27 million human deaths annually worldwide. Surface waters are impacted by anthropogenic factors, which contribute to the emergence and spread of ARB in the aquatic environment. While research on antibiotic resistance in surface waters has increased recently in developing nations, including Bangladesh, especially in aquaculture, such studies are still limited in Bangladesh compared to developed nations. In this study, bacteria strains isolated from three rivers and two lakes in Khulna city, Bangladesh were characterized for their antibiotic resistance using disk diffusion method. Of the 106 bacterial isolates from 180 surface water samples, the majority exhibited resistance to Ciprofloxacin (75.0-87.5%) and Ceftriaxone (65.6-78.1%), while resistance to Ampicillin was comparatively lower (9.4-18.8%). Notably, the prevalence of ARB was observed to be higher during the wet seasons compared to the dry seasons. The 16S rRNA gene analysis showed that Shigella flexneri was the most dominant (17.9%) bacterium among the ARB cultured from surface waters, followed by Escherichia fergusonii (12.5%), Proteus mirabilis (10.7%), and Enterobacter quasiroggenkampii (8.9%). At the genus level, Enterobacter (23.5%), Shigella (20.6%), and Escherichia spp. (14.7%) were the most abundant among the ARB in both river and lake samples. The findings of this study highlight the prevalence of antimicrobial resistance in surface water sources and suggest the need for enhanced monitoring and improved disposal practices to mitigate potential public health risks.