Chromosomal substitution lines of wheat variety Timstein (Tim) into the genetic background of variety Chinese Spring (CS) were used to evaluate the chromosomal location of genes controlling carbon isotope discrimination (Δ), photosynthesis rate (PR), stomatal conductance (SC), and grain yield. The experiment was carried out in the field at Shahrekord University Research Station. Considerable variation was observed among the substitution lines and between the parents for all traits. Chinese Spring had smaller values for the characters under study than Timstein. Significant correlations were found for grain yield with PR (r = 0.556) and Δ (r = 0.619). The substitution line CS (Tim2B) was significantly different from CS for Δ (P < 0.01). The substitution of homeologous group 3 chromosomes produced significant differences from the recipient variety for PR. Substitution lines CS (Tim3A), CS (Tim3B), CS (Tim4B), and CS (Tim1D) were significantly different from CS for SC. Genomic comparisons indicated that genome B had higher values of all four characters compared with the A and D genomes. Homeologous effects of genomes were documented for Δ and PR only and not for SC and grain yield.