Abstract: This study delves into the significance of the Rh (Rhesus) gene family in the high alkali adaptation of Gymnocypris przewalskii, conducting bioinformatics analysis on the genome data and exploring alkali stress responses through gene expression detection. The results showed that seven Rh gene family members encoding proteins ranging from 425 to 562 amino acids. Despite notable differences in gene structure, motif and domain analysis showcased a high level of conservation among these members. Chromosome mapping revealed the dispersion of Rh gene family members across seven chromosomes (Chr 37, 29, 46, 13, 14, 15, and 26), all encoding hydrophobic stable proteins, except for Rhd products, which were acidic proteins. The predominant alpha-helix secondary structure was observed in these proteins. Subcellular localization prediction positioned all Rh gene products within the cell membrane. Selection pressure analysis highlighted positive selection in Rhbg and Rhcg2a. Under carbonate alkalinity stress, Gymnocypris przewalskii exhibited a gradual increase in blood ammonia concentration, followed by a decline after 48h. Gene expression studies showed that the expression levels of Rhag, Rhbg, Rhcg1, Rhcg2a, and Rh50 in the gills, skin, kidney, and liver of Gymnocypris przewalskii increased initially and then decreased. In the blood tissue, the expression levels of Rhag, Rhbg, Rhcg2a, and Rh50 gradually decreased, while the expression level of Rhd first increased and then decreased. Rhbg and Rhcg2a may be the key genes involved in ammonia nitrogen metabolism in Gymnocypris przewalskii, while Rhag, Rhcg1, Rhcg2a, and Rh50 also playing significant role. In terms of tissues and organs, gills are primarily responsible for ammonia excretion followed by skin, kidney, and liver. This study revealed the role of RH gene family members in ammonia nitrogen metabolism of Gymnocypris przewalskii, and provided a scientific foundation for the conservation of this species.