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Phenotypic polymorphisms can be applied to study the micro-evolutionary forces that maintain genetic variation and can mediate speciation, but it can be difficult to determine the genetic basis of polymorphisms. Recently, restriction-site-associated DNA (RAD) sequencing has become popular, which can easily produce multiple single nucleotide polymorphisms from whole genomes. Here, we combined RAD sequencing, allele-specific PCR and Sanger sequencing to determine the genetic basis underlying male colour dimorphism of a Lake Tanganyika cichlid fish, Cyprichromis leptosoma. Our analyses using both a cross-family (two parents and 12 F2 males) and 64 wild individuals do not contradict a hypothesis that two alleles of one-locus control male colour dimorphism. Also, the locus may be located on a genome region that experiences reduced levels of recombination. Although more analyses will be needed to conclude these findings, this study is the first to suggest the genetic basis of a colour polymorphism using RAD sequencing., (© 2012 John Wiley & Sons Ltd.)