Chickpea (Cicer arietinum L.), a cool season crop is severely affected by heat stress, predicted to increase due to warming climates. Research for identifying heat tolerance markers for potential chickpea genotype selection is imperative. The study assessed the response of four chickpea genotypes to a natural temperature gradient in the field using chlorophyll fluorescence, non-structural carbohydrate, chlorophyll concentrations, gas exchange and grain yield. Field experiments were carried out in two winter seasons at three locations with known differences in temperature in NE South Africa. Results showed two genotypes were tolerant to heat stress with an Fv/Fm of 0.83-0.85 at the warmer site, while the two sensitive genotypes showed lower Fv/Fm of 0.78-0.80. Both dark-adapted Fv/Fm and Fq'/Fm' (where Fq' = Fm' -F) measured at comparable high light levels correlated positively with grain yield. The two tolerant genotypes also showed higher photosynthetic rates, starch, sucrose and grain yield than the sensitive genotypes at the warmer site. However, these parameters were consistently higher at the cooler sites than at the warmer. These results were further validated by a climate chamber experiment, where higher Fv/Fm decline in the sensitive compared to tolerant genotypes was observed when they were exposed to short-term heat treatments of 30/25 °C and 35/30 °C. Tolerant genotypes had higher Fv/Fm (0.78-0.81) and grain yield plant-1(1.12-2.37g) compared to sensitive genotypes (0.74-0.75) and (0.32-0.89g plant-1) respectively in the 35/30 °C. It is concluded that chlorophyll fluorescence and leaf carbohydrates are suitable tools for selection of heat tolerant chickpea genotypes under field conditions, while the coolest site showed favourable conditions for chickpea production.