The Tibetan Plateau, despite being the highest and least populated plateau on the Earth, harbors antibiotic-resistant bacteria (ARBs) as well as antibiotic resistance genes (ARGs) in its pristine environment and animals. There is, however, limited knowledge regarding antibiotic resistance in Tibetan Plateau animals. Here, fecal samples of grazing yaks and intestinal content samples of wild rats were collected at altitudes of 2920m and 4360m and subjected to selective culture, PCR, and metagenomic sequencing. The results revealed a higher antibiotic resistance rate in low-altitude regions than in high-altitude regions. The simultaneous presence of bacteria exhibiting different antibiotic resistance phenotypes across multiple samples suggests the potential emergence of multidrug-resistant strains within the animal gut microbiota. Pseudomonas spp. and Escherichia coli were the predominant ARBs. In contrast to low-altitude regions with frequent human activities, ARG dissemination in the Tibetan Plateau did not show a significant correlation with mobile genetic elements. ARGs in high-altitude regions relied more on vertical gene transmission. This study expands our knowledge of antibiotic resistance in grazing and wild animals inhabiting the Tibetan Plateau, thereby highlighting the potential threat posed by antibiotic resistance in this region.