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Pre-harvest sprouting (PHS) is one of the serious global issues in wheat production. Identification of quantitative trait loci (QTL) and closely-linked markers is greatly helpful for wheat improvement. In the present study, a recombinant inbred line (RIL) population derived from the cross of Zhongmai 578 (ZM578)/Jimai 22 (JM22) and parents were phenotyped in five environments and genotyped by the wheat 50 K single-nucleotide polymorphism (SNP) array. Two QTL of germination index (GI), QGI.caas3A and QGI.caas-5A, were detected, explaining 4.33%–5.58% and 4.43%–8.02% of the phenotypic variances, respectively. The resistant effect of QGI.caas-3A was contributed by JM22, whereas that of QGI.caas.5A was from ZM578. The two QTL did not correspond to any previously identified genes or genetic loci for PHSrelated traits according to their locations in the Chinese Spring reference genome, indicating that they are likely to be new loci for PHS resistance. Four kompetitive allele-specific PCR (KASP) markers K_AX109605367and K_AX-179559687 flanking QGI.caas-3A, and K_AX-111258240 and K_AX-109402944 flanking QGI.caas-5A, were developed and validated in a natural population of 100 wheat cultivars. The distribution frequency of resistance alleles at Qphs.caas-3A and Qphs.caas-5A loci were 82.7% and 57.1%, respectively, in the natural population. These findings provide new QTL and tightly linked KASP markers for improvement of PHS resistance in wheat. [ABSTRACT FROM AUTHOR]