Resistance to pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) is one of the most valuable traits in many breeding programs. However, the quantitative nature of inheritance of PHS resistance challenges the study of this trait. Near-isogenic lines (NILs) can turn a complicated quantitative trait into a Mendelian factor (qualitative) and are, therefore, valuable materials for identification of the gene(s) responsible for a specific phenotypic trait and for functional studies of specific loci. Five pairs of NILs were developed and confirmed for a major quantitative trait locus (QTL) located on the long arm of chromosome 3A contributing to PHS resistance in wheat. These NILs were generated by using the heterogeneous inbred family method and a fast generation-cycling system. Significant differences in PHS resistance between the isolines were detected in the NILs. The presence of the PHS-resistance allele from the resistant parent increased resistance to sprouting on spikes by 26.7-96.8%, with an average of 73.8%, and increased seed dormancy by 36.9-87.2%, with an average of 59.9% across the NILs. These NILs are being used for the identification of candidate genes responsible for this major PHS-resistance locus on wheat chromosome arm 3AL.