The cotton leafhopper, Amrasca biguttula (Ishida), a sucking insect pest of cotton and vegetables, is present throughout Southeast Asia. Patterns of molecular and morphological differentiation among A. biguttula populations collected from seven cotton growing areas of Punjab, Pakistan, were studied by sequencing the barcode region of the mitochondrial cytochrome oxidase subunit 1 (COI) gene and by morphometrically analyzing wing shape. Molecular analysis revealed absence of isolation by distance (r = 0.075, P = 0.341) in spite of significant population differentiation (overall F_ST = 0.15 ± 0.06 (SE)). Also, haplotype diversity varied between populations, and neutrality tests indicated an excess of rare alleles in one of the seven areas. Although leafhopper populations are clearly shaped by genetic drift and possibly also by founder events, wing shape and size were conserved across the study area. These results have important implications for understanding the processes affecting patterns of molecular and morphological variations of pest insects that often are strongly controlled by agrochemical applications.