The temperature data from 3 meteorological stations (Kashi, Ruoqiang, and Hotan) in the South of Tarim River Basin (STRB) during 1964–2011 were analyzed by Mann-Kendall test and correlation analysis. The results from Mann-Kendall test show that the surface temperature (ST), 850 hPa temperature (T850), and 700 hPa temperature (T700) exhibited upward trends, while 300 hPa temperature (T300) revealed a downward trend. On the whole, the change rate of ST, T850, T700, and T300 was 0.26~0.46°C/10a, 0.15~0.40°C/10a, 0.03~0.10°C/10a, and −0.38~−0.13°C/10a, respectively. For the periods, ST and T850 declined during 1964–1997 and then rose during 1998–2011. T700 declined during 1964–2005 and then rose during 2006–2011, while T300 rose from 1964 to 1970s and then declined. The results from correlation analysis show that T850 and T700 positively correlated with ST (P < 0.01) at the all three stations and there was a negative correlation between T300 and ST at Hotan (P < 0.1), while the correlation is not significant at Kashi and Ruoqiang. The results indicate that there were gradient differences in the response of upper-air temperature (UT) to ST change.