Spring wheat (Triticum aestivum L.) is one of the most imperative staple food crops with annual production of 765 million tons globally to feed ~ 40% world population. Studying agro-morphological traits and genetic diversity in available germplasm is crucial for sustainable wheat improvement to ensure global food security. A diversity panel of 184 Pakistani wheat genotypes was genotyped using 123,596 high quality based single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS) with 42% of the SNPs mapped on B, 36% on A and 22% on D sub-genomes of wheat. Chromosome 2B contains the most SNPs (9,126) and 4D has the least (2,660). Population structure analysis divided the population into two subpopulations. The mean polymorphic information content (PIC), genetic diversity (GD), and major allele frequency (MAF) of the population were 0.157, 0.1844 and 0.87, respectively. Analysis of molecular variance (AMOVA) revealed the higher genetic diversity (80%) within the subpopulation than among the subpopulations (20%). The genome-wide linkage disequilibrium (LD) decay was 3.38 cM. Among the three subgenomes, D has the highest LD decay value (7.45 cM), followed by A (2.88 cM) and B (2.03 cM). Results of this study will be a baseline to start marker-assisted selection (MSA), association mapping (AM), genome wide association studies (GWAS) and genome selection (GS) related breeding programs of wheat.Keywords. Genotyping by Sequencing (GBS), Genetic Diversity (GD), Population Structure (PS), Polymorphic Information Content (PIC), Major Allele Frequency (MAF).Participants: Shabbir Hussain1, Madiha Habib1, Amy Bernardo2, Paul St. Amand2, Guihua Bai2, Nida Ghori2, Azeem Iqbal Khan1, Faisal Saeed Awana1.1 Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad-38000, Pakistan;2 USDA, Hard Winter Wheat Genetics Research Unit, Manhattan, KS 66506, USA