Cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a serious pest of several crops throughout the world, representing millions of United States of America dollars worth of damage. This pest can adapt to various cropping systems in a wide geographical range and has high migratory potential. It features high fecundity and can develop resistance to almost all insecticides used for its management. Several investigations to develop microsatellite markers for H. armigera have not been successful because of the paucity of microsatellites in the lepidopteran genome. As well, collections of H. armigera from cotton fields of southern and western India were not yet studied for molecular genetic diversity. The current study aimed to screen publicly available expressed sequence tag resources for simple sequence repeats and assess their potential as DNA markers for assessment of gene flow between collections of southern and western India. We identified 30 polymorphic microsatellites for potential use in diversity analysis of H. armigera collections. Genetic diversity analysis revealed that the collections were widely diverse with population differentiation index (F
st) of 0.17. Furthermore, gene flow analysis revealed a mean frequency of private alleles of 11% within the collections. The microsatellite resources we developed could be widely used for molecular diversity or population genetic research involving this important pest of cotton and food crops.