Key message: Cytogenetic analysis and array-based SNP genotyping of wheat–Th. intermediumintrogression lines allowed identification of 634 chromosome-specific SNP markers across all twenty-one chromosomes ofTh. intermedium(StJ r J vs , 2n = 6x = 42). Abstract: Thinopyrum intermedium (2n = 6x = 42, StJ r J vs ) is one of the most promising reservoirs of useful genes including tolerance to abiotic stresses, perenniality and disease resistance not available in the cultivated bread wheat. The transfer of genetic diversity from wild species to wheat offers valuable responses to the effects of climate change. The new array-based single-nucleotide polymorphism (SNP) marker technology provides cheap and easy-to-use molecular markers for marker-assisted selection (MAS) in wheat breeding programmes. Here, we focus on the generation of a new chromosome-specific SNP marker set that can be used to characterize and identify the Th. intermedium chromosomes or chromosome segments transferred into wheat. A progressive investigation of marker development was conducted using 187 various newly developed wheat–Th. intermedium introgression lines and the Axiom ® Wheat-Relative Genotyping array. We employed molecular cytogenetic techniques to clarify the genome constitution of the Th. intermedium parental lines and validated 634 chromosome-specific SNPs. Our data confirmed the allohexaploid nature of Th. intermedium and demonstrated that the St genome-specific GISH signal and markers are present at the centromeric regions of chromosomes 1J vs , 2J vs , 3J vs and 7J vs . The SNP markers presented here will be introduced into current wheat improvement programmes, offering a significant speed-up in wheat breeding and making it possible to deal with the transfer of the full genetic potential of Th. intermedium into wheat.