The major histocompatibility complex (MHC) is a gene family common to vertebrates and is encoded by agroup of genes closely related to immunological functions. Many studies have indicated that MHC occurs on the surfaceof all karyocytes and that the MHC II genes of fish differ by species in structure, character, and polymorphisms.The spotted scat (Scatophagus argus) is eurythermic and euryhaline and is widely distributed in brackish water and marinehabitats of the Indo-Pacific region. Spotted scat has become an economically important species in southern Chinain recent years owing to its strong tolerance to a variety of environmental conditions. However, most spotted scat studiesin the last two decades have focused on artificial breeding, biological characteristics, gonadal development, reproductivecharacteristics, and genes related to osmotic adjustment and gonadal development, but not immune systemfunction. Furthermore, farmers have recently found that spotted scat are susceptible to infections by parasites, bacteria,and viruses, which could cause serious economic losses. Thus, we determined the complete cDNA sequence of thespotted scat MHC-IIβ gene for the first time using homology-based cloning and rapid amplification of cDNA ends polymerasechain reaction (RACE-PCR) and analyzed the introns, gene polymorphisms, and tissue expression. As results,the cDNA sequence of the spotted scat MHC-IIβ gene was 1172 bp in length, including a 34 bp 5′-untranslated region(UTR), a 388 bp 3′-UTR, and a 750 bp open reading frame (ORF). The ORF contained the signal peptide, the β1 domain,the β2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. The total length of the genewas 2920 bp. The MHC-IIβ gene contained six exons and five introns (85−1010 bp), and the third intron separated theβ2 domain into two parts. This structure differs from some other teleosts and provides new evidence for the lack ofconservation of MHC-IIβ gene structure. The molecular formula was C2239H3730N750O934S198, and relative molecularmass was 62.45 kD. Some classic functional sites were found in the amino acid sequence, such as a protein kinase Cphosphorylation site, an N-glycosylation site, a casein kinase II phosphorylation site, a tyrosine kinase phosphorylationsite, and an N-nutmeg acetoxylation site. Interestingly, the number of protein functional sites in the spotted scatMHC-IIβ gene was very similar to that in other fish, such as common carp, yellow croaker, and Japanese flounder, indicatingconservation of MHC in fish. We identified 48 alleles from 209 clones in 43 individuals to examine polymorphismsin the spotted scat MHC-IIβ gene. These alleles were named Scar-DXB*0101−Scar-DXB*4801, indicating highpolymorphism of the MHC-IIβ gene in S. argus. However, allelic polymorphisms of the MHC-IIβ gene are quite differentin fish, which may be an inherent characteristic of the species or it may be related to other factors, such as thefounder effect, gene flow, or gene selection during evolution. The RT-PCR analysis showed that the MHC-IIβ gene wasexpressed in all 11 tissues examined, with the highest levels in spleen, gill, intestine and skin; moderate levels in kidney,stomach, and heart; and low levels in the eyes, brain, liver, and muscle. MHC-IIβ gene mRNA expression levels variedin liver, spleen, gill, and kidney after infection with Aeromonas hydrophila, indicating that the gene has important effectson the immune response. A neighbor-joining tree showed that the S. argus MHC-IIβ gene had a fairly close geneticrelationship with those of Paralichthys olivaceus, Dicentrarchus labrax, and Salmo salar and more distant genetic relationshipswith Ginglymostoma cirratum, Gallus gallus, Mus musculus, and Homo sapiens. These results lay the foundationfor screening resistance genes and breeding S. argus for disease resistance.