Antibiotic resistance genes (ARGs) are emerging as environmental pollutants that can persist and disseminate in aquatic environments. Lakes, as important sources of freshwater, also serve as potential natural reservoirs of ARGs. In this study, we analyzed the distribution and potential risks of resistance genes in five typical freshwater lakes on the Yunnan-Guizhou Plateau. Our findings revealed that multidrug and MLS ARGs dominated in the studied lakes. Notably, while Lugu Lake exhibited higher abundance of ARGs, mobile genetic elements (MGEs), and metal resistance genes (MRGs), a greater resistome risk was observed in the eutrophic Xingyun Lake. The dissemination processes of ARGs and MRGs are primarily driven by microbial communities and the horizontal gene transfer via MGEs. Limnohabitans, Flavobacterium, and Acinetobacter were identified as key players in the dissemination of ARGs. Our study highlights the persistence of ARGs and provides valuable baseline data and risk assessment of ARGs in plateau freshwater lakes.