AbstractThe cloning of agriculturally important genes is often complicated by haplotype variation across crop cultivars. Access to pan-genome information greatly facilitates the assessment of structural variations and rapid candidate gene identification. Here, we identified the red glume 1 (Rg-B1) gene using association genetics and haplotype analyses in ten reference grade wheat genomes. Glume color is an important trait to characterize wheat cultivars. Red glumes are frequent among Central European spelt, a dominant wheat subspecies in Europe before the 20^th century. We used genotyping-by-sequencing to characterize a global diversity panel of 267 spelt accessions, which provided evidence for two independent introductions of spelt into Europe. A single region at the Rg-B1 locus on chromosome 1BS was associated with glume color in the diversity panel. Haplotype comparisons across ten high-quality wheat genomes revealed a MYB transcription factor as candidate gene. We found extensive haplotype variation across the ten cultivars, with a particular group of MYB alleles that was conserved in red glume wheat cultivars. Genetic mapping and transient infiltration experiments allowed us to validate this particular MYB transcription factor variants. Our study demonstrates the value of multiple high-quality genomes to rapidly resolve copy number and haplotype variations in regions controlling agriculturally important traits.