In this study, amplified fragment length polymorphism (AFLP) technique was used to analyze the genealogical relationship of 7 introgressed cotton varieties from the hybridization of upland (Gossypium hirsutum L) and sea-island cottons (Gossypium barbadense L). Ten pairs of primer combinations with high polymorphism and resolution were selected from 64 primers. These 10 primer combinations resulted in a total of 480 bands, of which 374 bands (77.9%) were polymorphic and 51 bands (10.6%) were specific. Cluster analyses showed that 7 varieties of cottons were divided into two groups. Our results suggested that interspecific hybridization is feasible to broaden germplasm of upland cotton and AFLP technique could be applied for the identification of variety purity and genealogical relationship.