This study was to determine phylogenetic relationships and genetic variation in Iranian Afshari sheep breed. For this reason, phylogenetic relationships and genetic variation were analyzed by using 12S rRNA and 16S rRNA gene sequences. The genomic DNA was isolated by salting out method and amplified 12S rRNA and 16S rRNA genes using PCR method. PCR amplification of 12S and 16S rRNA generated PCR amplicons at 859 and 1053 bp lengths, respectively. Sequence analysis was performed using Bio–Edit software. Phylogenetic tree was constructed using MEGA software. Phylogenetic analysis of haplotype in the combination with the sheep from Gen–Bank showed that Iranian Afshari sheep made a close to the Australian sheep cluster. There was found informative for establishing relationships between breeds from different parts of the world. This may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.