SummaryCanola varieties exhibit discernible variation in drought avoidance and drought escape traits, suggesting its adaptation to water-deficit environments. However, the underlying mechanisms are poorly understood.A doubled haploid (DH) population was analysed to identify QTL associated with water use efficiency (WUE) related traits. Based on the resequenced parental genome data, we developed sequence-capture based markers for fine mapping. mRNA-Seq was performed to determine the expression of candidate genes underlying QTL for carbon isotope discrimination (Δ13C).QTL contributing to main and QTL × Environment interaction effects for Δ13C and for agronomic WUE were identified. One multi-trait QTL for Δ13C, days to flower, plant height and seed yield was identified on chromosome A09, in the vicinity of ERECTA. Interestingly, this QTL region was overlapped with a homoeologous exchange event (HE), suggesting its association with the major QTL. Transcriptome analysis revealed several differentially expressed genes between parental lines, including in HE regions.This study provides insights into the complexity of WUE related genes in the context of canola adaptation to water-deficit conditions. Our results suggest that alleles for high Δ13C contribute positively to canola yield. Genetic and genomic resources developed herein could be utilised to make genetic gains for improving canola WUE.