Improving crop water-use efficiency (WUE) is critical in securing food production under arid and semiarid regions, but a quick and accurate method for quantifying crop WUE is lacking. A field study on winter wheat (Triticum aestivum L.) was conducted under six irrigation regimes in two growing seasons (2011–2012 and 2012– 2013) to determine the sampling time and the condition of soil moisture on the relationship of C isotope discrimination (13C; measured by flag leaves, other leaves, and kernel) with grain yield and WUE at different levels (leaf level as WUEL, biomass level as WUEb and grainyield level as WUEg). The results showed that the accumulated average soil water condition affected the leaf and kernel 13C. The kernel 13C and flag leaf 13C after heading were significantly related to the final biomass and grain production (P < 0.05), but their relationship to WUEg and WUEb was variable as a result of the effects of seasonal weather conditions on harvest index (HI) and soil evaporation. Kernel 13C or flag leaf 13C can be used to assess the yield of winter wheat but may not be able to predict the WUEg and WUEb. The WUEL was related to the soil water condition at the time when the measurements occurred. The WUEL might be a useful indicator of WUE at yield level when soil water condition at measuring of WUEL could represent the average soil water condition of the whole growing season of the crop.