Glass catfish (Kryptopterus vitreolus) are notable in the aquarium trade for their highly transparent body pattern. This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body, although certain black and silver pigments remain in the face and head. To date, however, the molecular mechanisms underlying this transparent phenotype remain largely unknown. To explore the genetic basis of this transparency, we constructed a chromosome-level haplotypic genome assembly for the glass catfish, encompassing 32 chromosomes and 23 344 protein-coding genes, using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines. Analysis revealed a premature stop codon in the putative albinism-relatedtyrp1bgene, encoding tyrosinase-related protein 1, rendering it a nonfunctional pseudogene. Notably, a synteny comparison with over 30 other fish species identified the loss of the endothelin-3 (edn3b) gene in the glass catfish genome. To investigate the role ofedn3b, we generatededn3b^−/− mutant zebrafish, which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish. These findings indicate thatedn3bloss contributes to the transparent phenotype of the glass catfish. Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish. These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish, but also offer a valuable genetic resource for further research on pigmentation in various animal species.