Abstract: The lack of genetic diversity is an obstacle for genetic improvement of upland cotton in China; thus, new technologies must be developed to produce more polymorphic molecular markers associated with agricultural traits of the existing resources for breeding. A highly efficient and economical technology of sequence-related amplified polymorphism (SRAP) molecular markers with an automated fragment analyzer ABI3500xl was developed, to detect genetic diversity in upland cotton. Using this new strategy, we easily screened for polymorphisms with 7,872 pairs of SRAP primers, and detected 504 polymorphic markers. Of all these, 165 were used for genetic diversity analysis in 128 upland cotton varieties collected nationwide in China. Our method combined a traditional molecular marker development technology with an economical and easy operation strategy for breeders.