Maize is among major field crops which provides food, fodder and various byproducts to the industry. Development of better performing varieties is very important to enhance and strengthen the maize production system. In this study ethyl methanesulfonate (EMS) is used to induce genetic variation in maize. Mutant population was derived from two genotypes 100,003 and 100,004. EMS was applied under three different concentrations of 25, 50 and 75 mM. 25 mM was found as an ideal concentration resulting in maximum survival rate. Total 10 SSRs were used in this study, which amplified 28 alleles with average of 2.7 alleles. Analysis of molecular variance showed significant differences present among individuals. Average heterozygosity for mutants derived from 100,003 and 100,004 was 0.58 and 0.53, respectively. UPGMA analysis characterized the mutants into two main and many sub clusters. According to the principal component analysis, PC 1 and 2 contributed to 64.2% variability with eigenvalue greater than 1. Statistics showed maximum coefficients of variance in traits of leaf area, cobb height and plant height. Promising mutants were also identified and recommended for future breeding programme. In conclusion, EMS mutagenesis is an effective technique to develop novel mutants that can be exploited in future breeding programmes.