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Understanding seasonal changes in soil microbial quotient (qMB, i.e., the MBC‐to‐SOC ratio) along altitudinal gradients and their dominant drivers is essential for determining the effects of environmental changes on the soil organic carbon pool. A 3‐year in situ soil core incubation experiment was thus conducted in a dry valley shrub, a valley‐mountain ecotone forest, a subalpine coniferous forest, an alpine coniferous forest, and an alpine meadow across a 2,431‐m altitudinal gradient on eastern the Qinghai‐Tibetan Plateau. Soil qMB in the soil organic layer (OL) and mineral soil layer (ML) was measured at five critical periods. The average soil qMB was significantly higher in the alpine meadow than at other sites, with no significant difference among the other sites. The average soil qMB in this region was less than the global average value (1.2%), indicating that the soil organic carbon pool of the local ecosystem has higher stability and a lower mineralization potential than that of the other ecosystems globally. Seasonal variations in soil qMB differed along the altitudinal gradient. Significant increases in soil qMB during the 3‐year incubation were mainly observed in the OL at higher altitudes, implying increased sensitivity of the soil organic carbon pool to environmental changes. Microbial biomass carbon and soil freeze‐thaw cycles were critical factors determining the altitudinal pattern and seasonal changes in soil qMB. Soil microbial biomass and freeze‐thaw regime changes under global climate change scenarios will alter the balance between SOC microbial assimilation and maintenance respiration demands, affecting soil carbon biogeochemical cycling in mountainous ecosystems. The highest soil microbial quotient (qMB) was observed in the alpine meadow, and slight differences were observed among other sitesSignificant increases in soil qMB during the 3‐year incubation were mainly observed in organic layer at higher altitudesThe microbial biomass carbon and soil freeze‐thaw cycle were critical factors determining the altitudinal pattern of and seasonal changes in soil qMB The highest soil microbial quotient (qMB) was observed in the alpine meadow, and slight differences were observed among other sites Significant increases in soil qMB during the 3‐year incubation were mainly observed in organic layer at higher altitudes The microbial biomass carbon and soil freeze‐thaw cycle were critical factors determining the altitudinal pattern of and seasonal changes in soil qMB