The temperature trend estimated from the distribution of bacterial branched glycerol dialkyl glycerol tetraethers (brGDGTs) in soil (i.e. the MBT–CBT or MBT′–CBT index) was studied along an altitude transect from 1850 m to 5139 m in the southeast Tibetan Plateau. Measured temperature values from weather stations in the area showed increasing seasonality with increasing elevation. The measured lapse rate of mean annual air temperature (MAAT) in the region was −7.2 °C/km, whereas that estimated from the MBT- or MBT′–CBT index was lower, with only −3.2 °C/km for the MBT′–CBT estimate. At elevation > 4 km, MBT′–CBT temperature values were increasingly higher than measured MAAT values. We hypothesized that the systematic changes in the length of the warm season, as well as the seasonal period of brGDGT production, with elevation are likely the cause of overestimation of the MBT′–CBT temperature at high elevation sites, and hence the underestimation of lapse rate. The hypothesis was supported by a similar lapse rate of −3.1 °C/km calculated from the measured mean warm season temperature (MWST) that only included monthly temperature values > 0 °C. This suggests that care should be taken in interpreting brGDGT derived temperature values in environments with large seasonal contrasts.