The LI-COR 6400 portable photosynthesis system is the most widely used and cited instrument for measuring leaf gas exchange in plant physiology. The focus of this paper is to evaluate three ways of computing/measuring leaf temperature: the LI-COR energy budget computation, the LI-COR leaf thermocouple (T/C), and an infra-red (IR) sensor chip (MLX90615) incorporated into the LI-6400 leaf chamber. The IR-sensor was calibrated against known targets, and then used to cross check the other two methods. The results showed that the IR-sensor was superior to the LI-COR thermocouple and agreed closer with the energy budget values of leaf temperature. The magnitudes of the errors were quantified in terms of °C difference in leaf temperature measured by T/C (as control) and IR sensor versus percentage difference in stomatal conductance (gs) and intercellular CO2 concentration (Ci) computed from leaf temperature. The temperature difference (IR versus other methods) in the range of -1.89 to 1.58 °C resulted in relative errors in gs from 14.7% to -18.0% and Ci from 16.5% to -7.8% of the values reported by the standard LI-6400 calculations depending on species and temperature. Reasons are given for trusting the IR-sensor over the other methods and it is suggested that the next generation LI-COR photosynthesis system include an IR-sensor.