Global climate change resulting in increased drought and higher ambient air temperatures may severely impact future productivity of the cotton industry. Identification of thermo-tolerant and water use efficient (WUE) cotton lines by CSIRO through plant breeding efforts may be utilized to maintain productivity despite unfavorable future climate extremes. Six cotton genotypes, which include DP16 (old genotype), Siokra L23 (WUE), CS50 (decreased WUE), 64224-212 (heat tolerant), SICALA V2 (poor heat tolerance) and Sicot 71, were grown in a sun-lit glasshouse under non-limiting water and nitrogen conditions at mid-day maximum air temperatures of 28 °C and 32 °C. We measured plant growth, photosynthetic capacity, Rubisco catalytic performance and online stable carbon isotope discrimination to calculate photosynthetic WUE and mesophyll conductance of CO2 (gm). Elevated growth temperature accelerated the onset of flowering and boll formation, and increased plant mass and total leaf area across all genotypes. Analysis of gas-exchange data indicates photosynthetic capacity was increased in all genotypes when measured at 32 °C compared to the identical lines at 28 °C, irrespective of growth temperature. Stomatal conductance (gs) measured under saturating light conditions varied across the genotypes with Siokra L23 displaying lowest gs resulting in improved instantaneous WUE. This was coupled with an improved gm hence CO2 assimilation remained similar to the other genotypes. In vitro Rubisco catalytic measurements at 25 °C indicated that cotton Rubisco has a high affinity for CO2 (KmCO2) and a slow kcat which was accompanied by a high specificity for CO2 as opposed to O2 (Sc/o). Analysis of Rubisco content revealed that Rubisco accumulates up to 45% of total leaf soluble protein indicating the significant investment of N into Rubisco synthesis. Therefore, future improvements in cotton photosynthesis could be achieved by improving Rubisco catalysis and reducing content to mitigate against the significant requirement for the large N investment into Rubisco.