Ovar-DRB1 is one of the most important response genes in the major histocompatibility complex (MHC) class II region of sheep. Gene polymorphism in the second exon of Ovar-DRB1 in three different Iranian fat-tailed sheep breeds (Lori-Bakhtiari, Shaul and Zandi) was analyzed by either restriction fragment length polymorphisms (RFLP) or direct sequencing. A total of 92 Lori-Bakhtiari, 40 Shaul, and 47 Zandi sheep were examined. PCR-RFLP identified 17 genotypes in Lori-Bakhtiari sheep, 12 in Shaul sheep and 11 in Zandi sheep. Collectively, 24 different genotypes could be found for Iranian fat-tailed sheep. Using direct sequencing, seven new sequences in exon 2 of the Ovar-DRB1 gene were identified. Generalized linear modeling with a multinomial error structure showed that the sheep populations had significantly different allele frequencies.