Barley (Hordeum vulgare L.) yield is commonly limited by low rainfall and high temperature during the growing season on the Canadian Prairies. Empirical knowledge suggests that carbon isotope discrimination (Δ(13)C), through its negative relationship with water-use efficiency (WUE), is a good index for selecting stable yielding crops in some rain-fed environments. Identification of quantitative trait loci (QTL) and linked markers for Δ(13)C will enhance its use efficiency in breeding programs. In the present study, two barley populations (W89001002003 × I60049 or W × I, six-row type, and Merit × H93174006 or M × H, two-row type), containing 200 and 127 recombinant inbred lines (RILs), were phenotyped for leaf Δ(13)C and agronomic traits under rain-fed environments in Alberta, Canada. A transgressive segregation pattern for leaf Δ(13)C was observed among RILs. The broad-sense heritability (H (2)) of leaf Δ(13)C was 0.8, and there was no significant interaction between genotype and environment for leaf Δ(13)C in the W × I RILs. A total of 12 QTL for leaf Δ(13)C were detected in the W × I RILs and 5 QTL in the M × H RILs. For the W × I RILs, a major QTL located on chromosome 3H near marker Bmag606 (9.3, 9.4 and 10.7 cM interval) was identified. This major QTL overlapped with several agronomic traits, with W89001002003 alleles favoring lower leaf Δ(13)C, increased plant height, and reduced leaf area index, grain yield, harvest index and days to maturity at this locus or loci. This major QTL and its associated marker, when validated, maybe useful in breeding programs aimed at improving WUE and yield stability of barley on the Canadian Prairies.