Abstract: The major histocompatibility complex (MHC) is crucial in immune function regulation and is a primary factor in immune responses within organisms. To explore the characteristics and roles of this gene in Gymnocypris przewalskii, the full-length cDNA sequences of MHCⅡ and chaperone gene Ii were obtained by cloning using rapid-amplification of cDNA ends (RACE) technique. Meanwhile, the sequences of the coding regions of MHCⅡ and Ii in G. eckloni were also obtained and compared. Sequence alignment and analysis revealed that the characteristics of respective genes were largely identical in both species. The construction of phylogenetic tree indicated the closer evolutionary position of MHCⅡ in both fish, clustered to one family, Cyprinidae. The amino acid sequences of MHCⅡα/β from two species both contained a signal peptide, two functional domains, a transmembrane domain, in which a conserved structure found in many different species located, and a cytoplasmic region. Quantitative real-time PCR (qRT-PCR) results showed that MHCⅡ was highly expressed in the kidney, brain, gill, liver, and skin tissue around the anal fin of G. przewalskii, while the expression of which displayed a higher level in the brain, muscle, eye, and gill of G. eckloni. The expression of MHCⅡ in gill, kidney, liver, and intestinal tissues was also compared between normal and Saprolegnia infected individuals of G. przewalskii, results demonstrated that the expression level of 3 genes in MHCⅡ was significantly down-regulated in the kidney tissues, while α, β genes were significantly up-regulated in gill, liver and intestinal tissues. MHCⅡ mRNA levels were significantly reduced in gill and kidney tissues after different saline stress treatments on G. przewalskii. Gene polymorphism analysis indicated that MHCⅡα has 8 and 12 allelic genotypes, encoding 23 and 24 amino acid sequences in two species, respectively, with sequence polymorphisms primarily in the α1 functional region. MHCⅡβ had 12 and 14 allelic genotypes, encoding 22 and 23 amino acid sequences in two species, respectively. The lower polymorphisms in MHCⅡ α/β of G. przewalskii suggested a weaker association with salinity tolerance compared to G. eckloni. Genetic diversity analysis showed high population diversity in both species, with haplotype diversity approaching 1 and minimal differences in nucleotide diversity. The slightly higher haplotype and nucleotide diversity of the MHCⅡ in G. eckloni indicated a somewhat greater population stability compared to G. przewalskii. Our research verified that MHCⅡ not only plays an important role in immunologic defence of two kinds of fish but also involved in the salinity tolerance process of G. przewalskii, while gene polymorphisms were not highly associated with salinity tolerance.