Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear 1–3. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy 4–8. Barley is a model crop 9, 10 and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H 11–19. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar “Azumamugi” (Az) and the non-dormant cultivar “Kanto Nakate Gold” (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. •Mitogen-activated Protein Kinase Kinase 3 (MKK3) regulates seed dormancy in barley•N260T substitution in the dormant allele decreases MKK3 kinase activity•The substitution appears to be causal for the seed dormancy QTL Qsd2-AK•The dormant allele is prevalent in barley cultivars grown in East Asia Seed dormancy affects germination and pre-harvest sprouting. Nakamura et al. report that the mitogen-activated protein kinase cascade participates in regulating barley seed dormancy. Their results suggest a natural mutation in MKK3 was used to improve pre-harvest sprouting tolerance in East Asia and may enable improvement of this trait in wheat.