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The Advanced Along-Track Scanning Radiometer (AATSR) land surface temperature (LST) product has a long-term time series of data from 20 May 2002 to 8 April 2012 and is a crucial dataset for global change studies. Accuracy and uncertainty assessment of satellite derived LST is important for its use in studying land–surface–atmosphere interactions. However, the validation of AATSR-derived LST products is scarce in China, especially in arid and semi-arid areas. In this study, we evaluated the accuracy of the AATSR LST product using ground-based measurements from 2007 to 2011 in the Heihe River Basin (HRB), China. The AATSR-derived LST results over Yingke site are closer to ground measurements than those over A’rou site for both daytime and nighttime temperatures. For nighttime, the averaged bias, STD, RMSE and R2 over both sites are 0.67 K, 3.03 K, 3.13 K and 0.93 K, respectively. Based on the accuracy assessment, we analyzed the AATSR-derived annual LST variations both in the HRB region and the two validation sites for the period of 2003 to 2011. The results at the A’rou site show an obvious increasing trend for daytime from 2003 to 2011. For the whole HRB region, the warming trend is clearly shown in the downstream of HRB.