The study was designed to evaluate the genetic background of indigenous germplasm by using simple sequence repeats (SSRs) gene markers for the selection of elite lines with multiple gene combinations that can be used in breeding programs for wheat improvement against rust disease. The principal component analysis (PCA) was constructed to estimate the genetic difference between the accessions and revealed an 85.3% variation. A high level of association was found between markers XWMC170 /XGWM608, XGWM44^a/XGWM44^b, XGDM111^a/XGWM129, XWMC765^a/XWMC765^b, and GWM148^a/XWMC765^b while low level of correlation was observed between the markers CSLV34b and CSLV34^a i.e -0.688 followed by GWM148^a and XGWM129 (-0.633). Cluster analysis revealed three main groups but group A was totally different from B and C with the genetic distance of 18.5 revealing that these accessions were having totally different genetic bases and are more diversified than the others. A total of 40 reported genetic markers (SSRs) were utilized to amplify the required bands for identifying the genes and their genetic distance in terms of variations between the wheat germplasm. The highest amplification was revealed by XGDM19 (130 bp), WMC773 (298 bp), XWMC331^a (210 bp), BARC114^a (105 bp), barc86^a (200 bp), and XBARC4^a (90 bp) i.e. 50, 48, 47, 46, 45, and 42 respectively. The study was useful as disease-related SSRs markers were used for general diversity, making the single SSRs marker used for multiple purposes including disease resistance, genetic diversity, and mineral or ion identification. It will ultimately help in saving the cost, time, and effort rather than using individual/or specific markers for evaluating the genetic distance or base for the diversity analysis.