Snakeskin gourami (Trichopodus pectoralis) is one of the most common air-breathing freshwater fish of the Indochina peninsula. It has a high meat yield and is one of the top five aquaculture freshwater fish in Thailand. However, it takes 2–3 years for adults to reach sexual maturity. Snakeskin gourami is not externally sexually dimorphic and its sex determination remains unknown, complicating many aspects of broodstock management including sex manipulation. Understanding the sex determination system will contribute significantly towards full-scale commercialization. By  characterizing the sex determination system in snakeskin gourami using cytogenetic approaches and Diversity Arrays Technology, we identified sex-specific loci in 16 phenotypic sex assignments of snakeskin gourami. Of the 39 loci present in all males, 4 male-linked loci reached the criteria of moderately sex-linked loci (70:30; males:females and 80:20; males:females). By contrast, only one female-linked locus was detected from moderately sex-linked loci. This suggests that snakeskin gourami exhibits an XX/XY sex determination mode. No different chromosomal patterns were observed in karyotype, C-banding, and microsatellite repeat fluorescence in situ hybridization mapping between males and females, and no male-specific loci of 100:0 (males:females) were observed in snakeskin gourami. This suggests that the putative Y chromosome is young and the non-recombination region is very cryptic. A total of 10.26% male-linked loci were involved with the sex developmental pathway in vertebrates and 5.13% showed partial homology with several amniote sex chromosomal linkages. Surprisingly, the hypothesis of an ancestral amniote super-sex chromosome with overlaps of partial sex chromosomal linkages was also found in teleosts. This approach provides a solid baseline to reveal the sex determination mechanism and identify potential sex determination regions in teleosts, allowing further investigation of genetic improvements in snakeskin gourami.