Spot blotch (SB) is a common foliar disease of barley, caused by the hemibiotrophic fungal pathogen Bipolaris sorokiniana. Predominately found in humid growing regions worldwide, the disease can cause yield losses of up to 30%. Genetic control through deployment of durable resistance in barley cultivars is the preferred, yet the majority of Australian cultivars are susceptible, despite the identification of major resistance loci in bi-parental studies. In this study, we explore the genetic architecture of spot blotch resistance in an Australian barley breeding program. Seedling (glasshouse) and adult (field) experiments using a single conidial isolate (SB61) were conducted across two consecutive years for 337 lines genotyped with 16,824 polymorphic DArT-seq™ markers. Two mapping approaches were investigated, a single-marker genome-wide association study (GWAS) and a haplotype-based local genomic estimated breeding values (GEBV) approach. Both methods detected two major regions on chromosomes 3H and 7H that contribute resistance across growth stages. Additional chromosomal regions on 1H, 3H and 6H were detected only in the local GEBV approach. Haplotype stacking revealed the importance of the 7H region for adult-plant resistance in combination with other resistance haplotypes, suggesting a significant gene by gene interaction and demonstrating the highly quantitative nature of the genetic control. Our study validates the presence of major spot blotch resistance loci in Australian breeding populations, while generating novel insight into the genetic architecture, and highlights the challenges and opportunities to improve resistance levels via haplotype stacking or whole-genome approaches.