BACKGROUND: Major histocompatibility complex (MHC) comprises a group of genes, which plays a central role in immune response. The exon 2 of BuLA-DRB3 is part of the MHC class II in buffalo that highly polymorphic, found to be associated with resistance/susceptibility to infections and also with production parameters. OBJECTIVES: The purpose of the present study is to identify BuLA-DRB3 polymorphism in Khuzestan buffaloes and compare this population with other Iranian and world buffalo populations. METHODS: Blood samples were taken from 136 unrelated Khuzestan river buffaloes. After DNA extraction, second exon of BuLA-DRB3 was amplified by the seminested PCR method. Then, the fragments produced by amplifying second exon were cut by RsaI restriction enzyme according to van Eijik method. In the following, allelic frequencies, genotype frequencies, expected and observed homozygosty and heterozygosity were calculated. RESULTS: In restriction fragment analysis 13 and 24 different allelic and genotype patterns were identified for RsaI restriction enzyme, respectively. 10 out of 13 alleles were previously reported. The most frequent genotype was oo)0.1691) and then followed by hh (0.1544) ,ll(0.1103), lw (0.0955), lh (0,0808), ha (0.0661) and lo (0.0514). Also four most frequent alleles were o (0.2721), h (0.2316),l (0.2316)and w (0.1176), respectively. These seven genotypes and four alleles form 72.76% and74.29% overall genotype and allele frequency of population.  In addition, estimation of heterozygosity/homozygosty and deviation from Hardy-Weinberg equilibriumofcorresponding population revealed observed homozygosty is more than heterozygosity and departure of population from Hardy-Weinberg equilibrium. CONCLUSIONS: The results indicated that exon 2 of the BuLA-DRB3 gene is highly polymorphic among Khuzestan buffaloes and although, there is differences between buffalo’s genetic polymorphism of distinct world regions, Khuzestan buffaloes’ population is similar to Egyptian buffaloes’ population.