PCR-GBS or multiplexing amplicon sequencing is an alternative method for reducing genome complexity with the use of selective primers and is an indispensable tool for plant breeding and genome research. In the research work, for the first time, a PCR-genotyping panel comprising 830 primers spread across the wheat genome (A, B and D) was developed. After filtration, the newly created panel consisted of 401 high quality markers. Among the three wheat genomes, the B genome had the highest (151 single nucleotide polymorphism (SNPs)), and the D genome (88 SNPs) had the lowest marker density. Out of 21 chromosomes, 1B and 3B were characterized with the highest, and chromosomes 3D and 4D with the least SNPs. The frequency of transition-type SNPs (84%) was significantly higher than transversion-type SNP markers (16%), with Ts/Tv ratio equal to 5.3. The reliability of the new genotyping panel was tested in the bread wheat (T. aestivum L.) collection. The genetic diversity coefficient and polymorphism information content for 69 bread wheat accessions were 0.252 and 0.205, respectively. The highest polymorphism was observed for var. ferrugineum və var. milturum, and the least variation was for var. hostianum. Using the PCoA analysis, 22.5% of the variations of the first three coordinates were elucidated. Thus, SNP markers included in the genotyping panel allowed identifying genotypes with unique profiles, as well as to generate a general picture of genetic diversity in the whole collection.