In this paper, the observation data of maximum frozen soil depth, air temperature and precipitation of 21 meteorological stations in the Three Rivers Source Region and the reanalysis data of European Center (EC) were used. The temporal and spatial distribution characteristics of seasonal maximum permafrost depth (MFSD) in Three Rivers Source Region were analyzed by means of rotating empirical orthogonal function (REOF), and the relationship between seasonal permafrost and climatic factors was also discussed. The results showed:(1) the spatial distribution of average MFSD in the Three Rivers Source Region in recent 40 years showed that Maduo station was the maximum, decreasing in all directions, the maximum value in the center was 218.85 cm, and the average MFSD in the whole region was 136.66 cm. (2) The spatial distribution of average variability in 40 years and the first mode time coefficient of REOF showed that MFSD in the Three Rivers Source Region presented an obvious downward trend in recent 40 years, the frozen soil layer was obviously thinner, and the average MFSD decline rate in the whole region was 0.51 cm/a. (3) Among the themal climatic factors, the humidity index, air temperature and precipitation were the more important climatic factors affecting the seasonal frozen soil in the Three Rivers Source Region. (4) The key area of seasonal frozen soil in the Three Rivers Source Region was in the northeast, including 7 meteorological stations. The typical high value years of MFSD were 1983, etc. and the typical low years were 1988, etc. Through the composite analysis of 500 hPa potential height field typical high value years and typical low value years, in the typical high (low) value years of seasonal frozen soil, 500 hPa potential height field showed the negative anomaly, the range of South Asia high was smaller (larger), the strength was weaker (stronger), the temperature at the center of the temperature field is lower (higher), and the seasonal frozen soil was thicker (thinner). The research results can provide meteorological support for the research, development and demonstration of eco-environmental protection technologies such as permafrost conservation, degraded wetland restoration, degraded grassland near-natural restoration, reveal the mechanism of soil-atmosphere interaction in the Three Rivers Source Region, deal with climate change, and provide a theoretical basis for the construction of Sanjiangyuan National Park.