We detected the deletion breakpoints of wheat ph1b mutant and the actual size of the deletion. Also， we developed ph1b deletion-specific markers useful for ph1b-mediated gene introgression and genome studies. The Ph1 (pairing homoeologous) locus has been considered a major genetic system for the diploidized meiotic behavior of the allopolyploid genome in wheat. It functions as a defense system against meiotic homoeologous pairing and recombination in polyploid wheat. A large deletion of the genomic region harboring Ph1 on the long arm of chromosome 5B (5BL) led to the ph1b mutant in hexaploid wheat 'Chinese Spring，' which has been widely used to induce meiotic homoeologous recombination for gene introgression from wild grasses into wheat. However， the breakpoints and physical size of the deletion remain undetermined. In the present study， we first anchored the ph1b deletion on 5BL by the high-throughput wheat 90K SNP assay and then delimited the deletion to a genomic region of 60，014，523 bp by chromosome walking. DNA marker and sequence analyses detected the nucleotide positions of the distal and proximal breakpoints (DB and PB) of the ph1b deletion and the deletion junction as well. This will facilitate understanding of the genomic region harboring the Ph1 locus in wheat. In addition， we developed user-friendly DNA markers specific for the ph1b deletion. These new ph1b deletion-specific markers will dramatically improve the efficacy of the ph1b mutant in the meiotic homoeologous recombination-based gene introgression and genome studies in wheat and its relatives.