Amplified fragmentlength polymorphism (AFLP) markers have been used to ascertainthe intensity of inherent diversity and relatedness in cotton (Gossypium spp.) plants. Theeffectiveness of this method to distinguish inter and intra specificdifference in cotton could be handy in cultivar recognition and in marker assistedparental selection tool for plant breeders. Twenty cotton cultivars belonging to Gossypium hirsutum L., and G. arborium L. from the Pakistan and US origin were used for AFLP based genetic diversity estimates. The objective of this study was to assess the level of genetic variation among some cotton cultivars belonging to the old and new world species of cotton. Four EcoRI-MseI primer-pair combinations were used forthe AFLP analysis. The AFLP data assigned the genotypesinto groups that corresponded with their origin and lineagerelationships and showed a narrow genetic base among these cultivars.