The extreme ecosystems of the Tibetan Plateau harbor an alarming reservoir of antibiotic resistance genes (ARGs) within wildlife gut microbiomes—an emerging contaminant with significant implications for One Health. However, seasonal dynamics and pathogenic risks of these ARGs remain poorly understood. Through a 2.5-year study comparing the subterranean plateau zokor (Eospalax baileyi) and the surface-dwelling plateau pika (Ochotona curzoniae), we demonstrate how species-specific ecological strategies shape distinct ARG transmission patterns. Our results demonstrated striking eco-evolutionary trade-offs: surface pikas showed 1.3 times higher ARG diversity with strong seasonal variation, influenced by temperature-modulated bacterial community turnover and mobile genetic elements (MGEs). Conversely, subterranean zokors maintained stable, were enriched with high-risk ARGs dominated by mepA and tetO, reflecting their isolated niche. Both species consistently carried pathogenic-ARG complexes (Clostridium-bacA, Staphylococcus-Ermb) across seasons, genders, and ages, indicating established resistance reservoirs in plateau food webs. Metagenomic binning revealed co-transfer potential between ARGs and MGEs. This pioneering longitudinal study exposes a dual pattern: seasonal changes cause temporary turnover of ARGs, which harbor lower resistance risk in surface-dwelling animals, while subterranean hosts retain stable, higher-risk resistance. These findings establish critical baselines for monitoring antimicrobial resistance in vulnerable ecosystems and underscore the need for integrated One Health strategies.