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Key message: QTL analysis identified 16 QTLs, grouped in eight loci on seven soybean chromosomes that were associated with carbon isotope ratio (δ13C) in a biparental recombinant inbred population. Drought is a major limitation to soybean yield, and the frequency of drought stress is likely to increase under future climatic scenarios. Water use efficiency (WUE) is associated with drought tolerance, and carbon isotope ratio (δ13C) is positively correlated with WUE. In this study, 196 F6-derived recombinant inbred lines from a cross of PI 416997 (high WUE) × PI 567201D (low WUE) were evaluated in four environments to identify genomic regions associated with δ13C. There were positive correlations of δ13C values between different environments (0.67 ≤ r ≤ 0.78). Genotype, environment, and genotype × environment interactions had significant effects on δ13C. Narrow sense heritability of δ13C was 90% when estimated across environments. There was a total of 16 QTLs on seven chromosomes with individual QTLs explaining between 2.5 and 29.9% of the phenotypic variation and with additive effects ranging from 0.07 to 0.22‰. These 16 QTLs likely identified eight loci based on their overlapping confidence intervals. Of these eight loci, two loci on chromosome 20 (Gm20) were detected in at least three environments and were considered as stable QTLs. Additive QTLs on Gm20 showed epistatic interactions with 10 QTLs present across nine chromosomes. Five QTLs were identified across environments and showed significant QTL × environment interactions. These findings demonstrate that additive QTLs and QTL × QTL interactions play significant roles in genetic control of the δ13C trait. Markers flanking identified QTLs may facilitate marker-assisted selection to accumulate desirable QTLs to improve WUE and drought tolerance in soybean. [ABSTRACT FROM AUTHOR]