Efficiently assessing drought resistance of different cultivars is crucial for genotypic selection. The existing indicators, however, do not adequately cover both aspects of drought resistance: grain yield differences among cultivars under drought conditions, and drought sensitivity of a specific cultivar in response to water condition changes from wet to dry. Carbon isotopes have shown promise in this regard, but the commonly used bulk leaf carbon isotope (813Cbulk) is not consistently correlated with grain yield, resulting in an inability to consistently predict drought resistance. This study explored the effectiveness of carbon isotopes, particularly the n-alkane carbon isotope (813Calk), in assessing drought resistance of different cultivars in both aspects of drought resistance and provide insights into the potential mechanisms behind the inconsistent relationships between 813Cbulk and grain yield. We conducted field experiments involving multiple cultivars and irrigation treatments on winter wheat (Triticum aestivum L.) and measured the 813Cbulk and 813Calk on various growth stages and organs. Our study found that neither leaf 813Cbulk nor 813Calk could consistently predict the grain yield difference among cultivars under the same water treatment. This inconsistency is due to the influence of cultivar characteristics and post-photosynthetic carbon isotope fractionations. However, leaf 813Calk demonstrated a more robust and consistent correlation than leaf 813Cbulk as a surrogate for the drought sensitivity of different cultivars to reduced water conditions. The accuracy of evaluation can be influenced by the sampling periods and organs, with the optimal strategy being the collection of flag leaf at early grain filling stage. The key findings of our study highlight the advantages of leaf 813Calk in assessing the crop drought sensitivity and evaluating drought-resistant cultivars.