SRAP (Sequence-related Amplified Polymorphism), a new marker system, was applied in cotton genome analysis. We developed an efficient PCR reaction system for detecting SRAP that showed reliable, effective and reproductive. SRAP marker was used to detect the polymorphisms between 'Pima90' (G. barbodense) and ' Handan208' (G. hirsutum) and showed comparatively high polymorphism. Among the 30 primer pairs, 29 pairs generated 149 polymorphic bands with an average of 5.14 polymorphic bands per primer pair. The primer pair showing most polymorphism was the combination of me3 and em2, which produced 13 polymorphic bands. The SRAP marker was also tested among 11 upland cotton cultivars, 15 of the tested 30 primer pairs showed polymorphism yielding 22 polymorphic genetic loci, which is relatively high among upland cotton cultivars. The results showed that SRAP marker could be widely used in molecular genetic map construction and germplasm evaluation of cotton.