Core Ideas The 13C/12C ratio (δ13C) for tissues at bloom were closely associated with δ13C in mature seed. Repeatability of δ13C from tissues sampled at bloom and mature seed ranged from 0.79 to 0.94. Genotype × environment × tissue for δ13C was not significant. Genotypic ranking of seed δ13C was similar to other tissues, simplifying its use in phenotyping. Carbon isotope ratio (δ13C) has been used as a proxy for water use efficiency (WUE) in several crops. We compared δ13C of soybean [Glycine max (L.) Merr.] tissues sampled at bloom with δ13C of mature seed (MS). Twenty Maturity Group IV accessions with contrasting δ13C values were evaluated in three environments. Genotype and environment effects on δ13C were significant for the uppermost mature trifoliolate leaf, center leaflets from main stems, whole shoot, and MS, but there were no significant genotype by environment interactions, indicating that genotype rankings were similar among environments. Repeatability of δ13C among tissues ranged from 0.79 to 0.94, and MS δ13C values were significantly correlated with δ13C from other tissues (r ≥ 0.69). For genotypes of similar maturity, MS δ13C may be used as a selection tool for WUE, simplifying the use of δ13C for high-throughput phenotyping.