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Insufficient moisture availability often limits soybean [Glycine max (L.) Merr.] yield. Carbon isotope ratio (δ13C) provides an integrated measure of water use efficiency in C3 plants due to its substantial genetic variance, high heritability, and small genotype × environment interaction (G × E). The objective of this study was to identify quantitative trait loci (QTLs) associated with δ13C using a recombinant inbred line population derived from a cross between 'KS4895' and 'Jackson'. The field experiment was conducted in five environments to evaluate δ13C under rainfed and irrigated conditions. Analysis of variance of δ13C averaged over environment and irrigation treatment showed significant effects of genotype (G), environment (E), and G × E interactions. Heritability of δ13C in different environments and irrigation treatments ranged from 66 to 79%. Averaged over environments and irrigation treatments, heritability was 83%. A total of 24 QTLs associated with δ13C were identified and clustered in nine genomic regions on seven chromosomes. The QTL clusters on Gm05 (1), Gm06 (2) and Gm20 (1) were detected across different environments and irrigation regimes. Collectively, these four QTL clusters accounted for 55% of the phenotypic variation in δ13C. The QTLs on Gm06 and Gm20 also showed additive × additive epistasis that contributed approximately 4.2% to the total phenotypic variation. Several identified δ13C QTLs overlapped with QTLs associated with other physiological traits related to plant water status, biological nitrogen fixation, and plant morphology. The identified genomic regions may be an important resource in genomic selection studies to improve drought tolerance in soybean. [ABSTRACT FROM AUTHOR]