As an important biomarker, fatty acids (FAs) have been extensively used to trace the origin of organic matter in sediments and soils. However, studies of the distribution and abundance of FAs in alpine grassland soils are still rare, especially on the Qinghai-Tibetan Plateau (QTP), the highest plateau in the world, which contributes sediments to many large rivers in Asia. This study investigates the composition, distribution and source of FAs with increasing soil depths from 17 typical alpine grassland sites in the QTP. The most abundant FAs included the ubiquitous C16 FA and even-numbered long-chain FAs (C20–C30), indicating mixed inputs from microbial and higher plant sources. Source apportionment showed that higher plants were the dominant contributor of FAs (approximately 40%) in QTP soils. The abundance of FAs decreased with soil depth, with the highest value (1.08±0.09 mg/g OC) at a 0–10 cm depth and the lowest value (0.46±0.12 mg/g OC) at a 50–70 cm depth, due to much lower plant inputs into the deeper horizons. The total concentration of FAs was negatively correlated to the mean annual temperature (MAT; P<0.05) and soil pH (P<0.01), suggesting that the preservation of FAs was favored in low-MAT and low-pH soils on the QTP. The abundance of fresh OC source FAs increased significantly with the mean annual precipitation (MAP; P<0.05), indicating that high MAP facilitates the accumulation of fresh FAs in QTP soils. Other environmental parameters, such as the soil mineral content (aluminum and iron oxide), microbial community composition as well as litter quality and quantity, may also exert a strong control on the preservation of FAs in QTP soils and warrant further research to better understand the mechanisms responsible for the preservation of FAs in QTP soils.