Bread wheat (Triticum aestivum L.) is among the top three cereal crops and major staple food as it accounts for 20% calories, consumed by humans in the world. The vulnerability of wheat to diseases is a risk to the global food security. Three types of rusts, caused by fungal strains, are the major yield limiting factors to reduce overall wheat production. In the present study, molecular markers were employed for genetic diversity analysis of wheat accessions regarding the presence of various rust resistance genes. Three hundred and sixty wheat accessions were screened against stem rust (Sr), leaf rust (Lr) and yellow rust (Yr) resistance genes. Eleven polymorphic SSRs detected 20 alleles, ranging from two to four alleles per locus. The allele frequency depicted that average number of alleles per locus was 2.55, 2.82, 2.82, 2.64 and 2.82 for population A, B, C, D and overall respectively. Gene diversity and heterozygosity were high for locus Sr25 but its major allele frequency was low. The PIC value was high at loci Lr34 and Yr18. Gene diversity, heterozygosity and PIC value were the lowest for locus Sr39 but its major allele frequency was the highest. Gene diversity increased with the number of alleles, that had a low major allele frequency at a particular locus. The Polymorphic loci effectively discriminated 360 accessions into six major clusters via principal component and population structure analyses. Significant deviation of FST from zero in six suggested populations for seven loci indicated population differentiation and limited gene flow among them. A reduced median network was established, which suggested that taxon G54 (MaxiPak-65), G-42 (Chanab-70), G-66 (SA-42) and G-75 (Yecora-70) are the progenitors, which are sharing rust genes to Pakistani wheat. These SSRs appeared effective for estimating genetic diversity, population structure and gene flow for rust resistance and for marker assisted wheat breeding.