Maruca vitrata (Fabricius) is an invasive insect pest capable of causing enormous economic losses to a broad spectrum of leguminous crops. Microsatellites are valuable molecular markers for population genetic studies; however, an inadequate number of M. vitrata microsatellite loci are available to carry out population association studies. Thus, we utilized this insect's public domain databases for mining expressed sequence tags (EST)-derived microsatellite markers. In total, 234 microsatellite markers were identified from 10053 unigenes. We discovered that trinucleotide repeats were the most predominant microsatellite motifs (61.53%), followed by dinucleotide repeats (23.50%) and tetranucleotide repeats (14.95%). Based on the analysis, twenty-five markers were selected for validation in M. vitrata populations collected from various regions of India. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) ranged from 2 to 5; 0.00 to 0.80; and 0.10 to 0.69, respectively. The polymorphic loci showed polymorphism information content (PIC), ranging from 0.09 to 0.72. Based on the genetic distance matrix, the unrooted neighbor-joining dendrogram differentiated the selected populations into two discrete groups. The SSR markers developed and validated in this study will be helpful in population-level investigations of M. vitrata to understand the gene flow, demography, dispersal patterns, biotype differentiation, and host dynamics.