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Simple Summary: Strong ultraviolet radiation is an extreme environmental characteristic of the Tibetan Plateau. Melanin is the most important light-protective substance that allows fish to resist ultraviolet radiation. In order to explore the melanin protection mechanism of fish in the Tibetan Plateau against strong ultraviolet radiation, Triplophysa stenura distributed in high elevation areas was selected as the research object. Transcriptome differences were compared between skin and peritoneal wall layers with different melanin contents to explore the regulatory genes related to melanin synthesis. This provides basic data for analyzing the molecular mechanism of melanin protection in plateau fish against strong ultraviolet radiation. The results indicate that a total of twenty-three DEGs are enriched in the melanin synthesis pathway by a local Blast comparison, of which nine DEGs are significantly upregulated in the peritoneal wall layer and six DEGs are significantly upregulated in the dorsal and lateral skin. The results suggest that these genes may be associated with the molecular mechanism of melanin synthesis in T. stenura, and the differential regulation of genes may be related to the differences in the UVR intensity and tissue sites of melanin synthesis. Further investigation is needed to determine how these genes specifically regulate melanin synthesis. A total of 81,868 All-Unigenes were sequenced and assembled by the transcriptome in the dorsal skin, the lateral skin, and the peritoneal wall layer of Triplophysa stenura with a total assembly length of 123,827,585 bp, and 68,750 unigenes were annotated to seven functional databases. A total of 588 DEGs were screened between the dorsal and lateral skin, 17,097 DEGs were screened between the dorsal skin and the peritoneal wall layer, and 16,598 DEGs were screened between the lateral skin and the peritoneal wall layer. Most of DEGs in three tissues were annotated to GO terms related to cellular structures, binding, cellular processes, and catalytic activity. They were also annotated to KEGG pathways such as the MAPK signaling pathway, PI3K-Akt signaling pathway, Wnt signaling pathway, melanogenesis, tyrosine metabolism, and cell cycle. A total of twenty-three DEGs were found to be enriched in the melanin synthesis pathway by a local Blast comparison, of which nine DEGs were significantly upregulated in the peritoneal wall layer and six DEGs were significantly upregulated in the dorsal and lateral skin. The results suggest that these genes may be associated with the molecular mechanism of melanin synthesis in T. stenura, and the differential regulation of genes may be related to the differences in UVR intensity and tissue sites of melanin synthesis. Further investigation is needed on how these genes specifically regulate melanin synthesis. [ABSTRACT FROM AUTHOR]