Photosynthetic efficiency has become the target of several breeding programs since the positive correlation between photosynthetic rate and yield in soybean suggests that the improvement of photosynthetic efficiency may be a promising target for new yield gains. However, studies on combining ability of soybean genotypes for physiological traits are still scarce in the literature. The objective of this study was to estimate the combining ability of soybean genotypes based on F2 generation aiming to identify superior parents and segregating populations for physiological traits. Twenty-eight F2 populations resulting from partial diallel crossings between eleven lines were evaluated in two crop seasons for the physiological traits: photosynthesis, stomatal conductance, internal CO2 concentration, and transpiration. General combining ability (GCA) of the parents and specific combining ability (SCA) of the F2 populations were estimated. Our findings reveal the predominance of additive effects in controlling the traits. The genotype TMG 7062 IPRO is the most promising parent for programs aiming at photosynthetic efficiency. We have also identified other promising parents and proposed cross-breeding with higher potential for obtaining superior lines for photosynthetic efficiency.