Lygus hesperus (Hemiptera: Miridae) is the dominant species of Lygus bug complex in Texas cotton. However, there exists a considerable variation in L. hesperus populations within a narrow geographic range in west Texas. Genetic characterization of Lygus populations is a necessary first step in understanding Lygus population dynamics, movement, migration, development of pesticide resistance, evolution, speciation, and other ecological processes. Simple sequence repeat (SSR) or microsatellite markers are widely used as markers in insect ecology due to its genome-wide availability, reproducibility and high polymorphism. Inter simple sequence repeat (ISSR) marker is another potential alternative which is easier to develop compared with that of SSR. The primary objective of this study was to evaluate the application of recently developed 15 polymorphic SSR markers in genetic diversity study of Lygus hesperus from different cotton growing regions in Texas. We also compared the suitability of SSR and ISSR markers in phylogeographic study of L. hesperus. The PCR, vertical polyacrylamide gel electrophoresis, and fast silver staining (15 min) conditions were optimized for high throughput and less expensive procedure of genotyping (400 samples per day per person) using mega gel system. Eighty adult L. hesperus samples per population from five locations were genotyped using both SSR and ISSR markers. Results from both SSR and ISSR data analyses and comparative advantage of each marker in genetic diversity study will be discussed.