Sex reversal in insects, amphibians, reptiles, and fishes is a complicated and interesting biological phenomenon. Sex reversal changes the sex ratio of populations and may complicate breeding schemes. In the Chinese tongue sole (Cynoglossus semilaevis), genetic females may change into pseudomales, thereby increasing aquaculture costs because of the lower growth rate of the males than that of the females. Here, we identify a new locus associated with sex reversal; this single nucleotide polymorphism (SNP) is located in the third intron of the doublesex and mab-3 related transcription factor 1 (Dmrt1) gene on the Z chromosome (named Cyn_Z_8564889) and has two alleles, A and G. Cyn_Z_8564889 regulates sex reversal interactively with our previously detected SNP (Cyn_Z_6676874), with the genetic females simultaneously carrying the T allele of Cyn_Z_6676874 and the A allele of Cyn_Z_8564889 changing into pseudomales. Other Dmrt1 polymorphisms were detected, which formed two haplotypes. Two SNPs in the second exon of Dmrt1 result in amino acid changes, suggesting that Dmrt1 is essential in sex reversal. We also verified that pseudomales produce no or little W sperm. The interaction and linkage between Cyn_Z_6676874 and Cyn_Z_8564889 and the absence of W sperm from pseudomales unravel the genetic architecture of sex reversal in C. semilaevis.