Bighead catfish (Clarias macrocephalus, Günther, 1864) is an important aquacultural species that plays a crucial role in the economy of Southeast Asia. Crossbreeding between female bighead catfish and male African catfish (C. gariepinus, Burchell, 1822) is used to produce hybrids with vigorous phenotypes. However, sterility of the hybrid is a major obstacle to their mass production. There is an emerging hypothesis that the complexity of the sex-determination system between two parental species might affect sterility. Previous studies investigated the co-existence of XX/XY and ZZ/ZW sex-determination systems in the African catfish population in Thailand, but in bighead catfish the sex-determination system remains poorly understood. In this study, the sex-determination system of the bighead catfish was examined using Diversity Arrays Technology to identify the genomic variants associated with sex-linked regions. The results support the hypothesis of the previous study that the bighead catfish might exhibit a male heterogametic XX/XY sex-determination system with multiple male-linked loci. One of the male-linked loci showed homology with the GTSF1L gene, which shows a testis-enriched expression pattern. Two of the male-linked loci were partially homologous to transposable element. Male-linked loci on the putative Y sex chromosome were identified as an extremely small proportion of the genome. A PCR-based DNA marker was developed to validate the male-linked loci in the bighead catfish. Our findings provide novel insights into sex-determination mechanisms in clariid catfish and will contribute to genetic improvements in breeding programs.