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Carbon isotope discrimination (CID) has been suggested as an indirect selection criterion for grain yield under drought stress in wheat (Triticum aestivum L.). However, the association between CID and grain yield varies greatly among different studies. In this study, conducted in western Kansas, a temperate semi-arid region, the genetic variation of grain CID and its association with grain yield and other agronomic traits were examined using 43 preliminary and 15 advanced breeding lines under dryland conditions. The 43 preliminary breeding lines were tested in three environments while the 15 advanced breeding lines were tested in one environment. Results showed that both preliminary and advanced breeding lines had significant genetic variations and relatively small coefficients of variation in grain CID, indicating grain CID as a promising trait for selection in wheat breeding programs. In the trials for the preliminary breeding lines, the association between grain CID and grain yield was significant (P < 0.05) and positive in all three environments with Pearson correlation coefficients ranging from 0.34 to 0.74. This correlation was stronger in a trial with expected post-anthesis drought stress. However, there was only a weak positive correlation between grain CID and grain yield in the trial for the advanced breeding lines. Winter injury may have confounded the grain CID and yield relationship. This study also revealed a significant (P < 0.05) and negative correlation between grain CID and grain protein content in all four trials with Pearson correlation coefficients ranging from -0.43 to -0.65, suggesting a possible impact on baking quality while selecting high grain CID. Therefore, our results suggest that grain CID could be useful for grain yield prediction in semi-arid areas with moderate drought stress. However, precaution should be taken for selecting grain CID because of the effect of environment on its association with grain yield and its negative correlation with protein content.