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Soybean (Glycine max (L.) Merr.) meets a large portion of its nitrogen (N) need via biological N2 fixation, which is highly sensitive to drought stress. Nitrogen isotope ratios between 15N and 14N (δ15N) can be used as a metric for relative differences among soybean genotypes for N2 fixation, as δ15N is negatively associated with N2 fixation. This study aimed to dissect the genetic basis of δ15N using a mapping population of 196 F6-derived recombinant inbred lines developed from a cross between PI 416997 and PI 567201D that was evaluated in multiple environments. There was a wide range of δ15N in all environments and narrow-sense heritability for δ15N was 35% when estimated across environments. Analysis of variance of δ15N showed significant effects of genotype and environment, whereas the genotype × environment interaction was not significant (P < 0.05). Inclusive composite interval mapping for individual environments identified 10 additive QTLs on seven chromosomes with additive effects ranging from 0.02 to 0.13‰ and that individually explained phenotypic variations from 1.72 to 9.34%. In total, eight QTL × environment interactions were found, and several genomic regions were involved in QTL × QTL interactions that were not identified as additive QTLs. These identified QTLs were co-localized with genomic regions associated with N2 fixation and other physiological traits identified in previous studies. A search for candidate genes resulted in detection of genes for nodulation and N-metabolism underlying many additive and epistatic QTLs. These identified regions may serve as potential targets for enhancing N2 fixation in soybean. [ABSTRACT FROM AUTHOR]