Pre-harvest sprouting (PHS) in bread wheat (Triticum aestivum L.) is one of the major abiotic constraints influencing production of high quality grain. Selection for pre-harvest sprouting (PHS) resistance in bread wheat (Triticum aestivum L.) in early generations is difficult because it is expressed as a quantitatively inherited trait and subject to environmental effects. The objectives of this study were to validate a major quantitative trait locus (QTL) for PHS resistance on chromosome 4A in bread wheat and to isolate near-isogenic lines for this QTL using marker-assisted selection. A total of 60 Canadian wheat cultivars and experimental lines were screened with three SSR markers in a QTL region for PHS resistance. The SSR markers DuPw004, barc170 and wmc650 explained 67%, 75% and 60% of total variation in germination (%), respectively, among different wheat genotypes. Marker assisted back crossing with DuPw004 reduced the population size in BC1F1 and BC2F1 generation by 41% and 59%, respectively. A survey of pedigrees of different genotypes revealed that the parental line RL4137 is a major source of increased PHS resistance in a number of western Canadian wheat cultivars. Microsatellite markers (DuPw004, barc170 and wmc650) will be useful for plant breeders to pyramid QTL from different PHS resistance sources.