Monthly hydrological and daily meteorological data were collected across the Three-Rivers Headwater Region (TRHR) over a period from 1956 to 2012. Modified Mann–Kendall tests, flow duration curves, and correlation statistics were performed to identify long-term trend and interrelationships between these hydro-meteorological variables and to analyse the factors influencing runoff. The results of these analyses are as follows. (1) In the last 57 years, the annual runoff in the Lancang River Basin (LRB) and the Yangtze River Basin (YARB) has shown an increasing trend, while the runoff in the main stream of the Yellow River Basin (YRB) was slightly reduced. (2) In the YRB and the YARB, both the high and low flows decreased and increased together, respectively, whereas in the LRB, the high flow decreased while the low flow increased. (3) In the TRHR, the proportional change in annual runoff due to climate variability accounted for >85% of the observed change, while anthropogenic activity and glacier melting was responsible for ∼15%. The contribution of anthropogenic activity in the YRB and LRB was higher than that in YARB due to the greater anthropogenic activity. The contribution of glacier melting in the YARB and LRB were obviously higher than that in YRB due to the higher densities of glaciers.