Single nucleotide polymorphisms (SNPs) are high-density natural sequence variations in genomes. They are considered to be the major genetic source of phenotypic variability within a given species and serve as excellent genetic markers. SNPs are useful in identifying candidate genes that contribute to disease and phenotypic traits. In non-model organisms, the application of SNPs has been limited, because of the expense and technical difficulties entailed in currently available SNP isolation techniques. In the present study, we have developed a rapid and effective method to isolate SNPs throughout the genome randomly. The DNA fragments containing SNPs could be isolated efficiently from background DNA. We analyzed ten isolated DNA fragments with this method in half-smooth tongue sole (Cynoglossus semilaevis)--a newly exploited and commercially important cultured marine flatfish in China--and found that nine of the fragments contained SNPs. The findings were confirmed successfully in different individuals. The method presented here is cost-effective and applicable to essentially any organism.