Sex-specific markers provide significant molecular basis for sex control breeding biotechnology to produce all-male or all-female fish in commercial breeding. Redtail catfish (Mystus wyckioides), one of the commercial bagrid catfishes distributed in Southeast Asian, which have a long sexual maturation period that can last 3-5 years and males have apparent growth advantage over females, but its sex determination system remains unknown. In this study, we first applied 2b-RAD-seq approach to identify three male-specific 2b-RAD-tags and one male heterogametic SNP locus and validated by blast to the genome survey sequences and PCR amplification in both wild and breeding populations. To get longer sex-specific region, we performed genome walking and obtained a 4,630 bp of Y-specific sequence and 4,581 bp of X-specific sequence from the 2b-RAD-tag ref189950 with 92.19% nucleotide identity between them. And 9,923 bp/3,935 bp of Y-specific sequences and 8,491 bp/5,172 bp of X-specific sequences were also identified with 77.49% and 57.07% nucleotide identity in ref208528 and ref210837, respectively. Subsequently, three different kinds of sex-specific primers with different length products were designed based on the detected highly sex differentiated regions and could be used to distinguish males and females both in wild and artificially bred populations. What is more, the X-specific fragment was discovered to produce the dosage effect association in females and in males. The data suggest that male heterogametic XX/XY sex determination system should exist in the redtail catfish. More significantly, the sex-specific markers are of great value to protect wild resources and improve the efficiency of all-male breeding practices for aquaculture in the redtail catfish.