Gas exchange analysis was used to characterize photosynthetic and stomatal responses to key environmental stimuli in five commercial lines of Pima cotton (Gossypium barbadense L.) which represent a gradient of selection for higher yields and heat resistance, and a primitive, uncultivated G. barbadense. At constant light and vapor pressure deficit, stomatal conductance increased linearly with air temperature in the 23 to 36$C range in all five commercial lines, and conductance at each temperature increased as a function of selection. In contrast, photosynthetic rates had a low sensitivity to temperature in the 23 to 36$C range, particularly in the advanced lines. In a segregating F2 population from a cross between the advanced line, Pima S‐6, and the primitive cotton, B368, the slope of the stomatal response to temperature in each F2 plant was positively correlated with the stomatal conductance measured at 40$C. An analysis of the frequency distribution of stomatal conductance in F1 and F2 progeny of the cross showed that the differences in stomatal conductance between lines were genetically determined. These data indicate that selection for higher yield and heat resistance in Pima cotton has caused genetically determined changes in stomatal properties. Characterization of the relationship between the altered stomatal properties and the attained increases in heat resistance and yields could make it possible to use these physiological traits as selection criteria in future breeding programs.