Simple sequence repeat (SSR) markers are widely used in plant genetics and breeding. However, there is very little SSR polymorphism in cotton. Traditional SSR genotyping methods are all analyses of product size. This leaves many markers undetected, lowering the utilization efficiency of SSRs. In the present study, during map construction, monomorphic SSRs were subjected to single-strand conformation polymorphism (SSCP) analysis to reveal polymorphism. Of the 4194 monomorphic SSR primers from the two mapping parents, 158 primers (3.77%) showed polymorphism and generated 174 polymorphic loci. Sequence analysis showed that the differences in PCR products between the mapping parent were solely due to base transition or transversion, which was in accordance with SSCP principles. For example, the difference between HAU1122 products by mapping parent was only a transversion from C to G. Genetic mapping showed 160 loci to have been integrated into the interspecific BC1 linkage map, 5 of them within QTLs related to cotton fiber quality. The present research enables some monomorphic SSRs to exhibit polymorphism and increases the utilization efficiency of the existing SSR primers. In addition, polymorphisms of some SSRs revealed by SSCP are important functional genes, which is of significance for comparative genomics and functional genomics.