Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait loci (QTL) revealed a major PHS QTL located on chromosome 2B.1 that was significant in 16 environments and explained from 5 to 31% of the phenotypic variation. The objective of this project was to fine map the PHS QTL interval on 2B.1. For fine mapping the QTL interval, ESTs (expressed sequence tags) and comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B but only 10 were located in the QTL interval. Recombinant backcross populations (BC1F4 and BC1F5) were developed by backcrossing selected double haploids to a recurrent parent and selfing to the F4 and F5 generations. In each generation, three markers in the PHS QTL interval were used to screen for recombinants. Comparative analysis revealed good macrocollinearity between the PHS interval and a 3 million base pair (mb) region in rice chromosomes 7 and 3, and a 2.5 mb region in Brachypodium Super_0. Fine mapping revealed that the 2B.1 PHS QTL interval contained 2 PHS QTLs. The first PHS QTL, located between Wmc453c and Barc55, contributed one third of phenotypic variation and collocated with the seed dormancy QTL. The second PHS QTL, between Wmc474 and rCaPK, contributed two thirds of the variation. The PHS resistance alleles were contributed from Cayuga parent. One of the PHS Cayuga resistance alleles originated in Golden Chief, a parent of Clark’s Cream. One of the candidate genes, Calmodulin/Ca2+ dependent protein kinase, linked with one PHS QTL. Although many recombinant families were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.