BACKGROUND:Avian reoviruses (ARVs) are members of theOrthoreovirus genus; one of the 12 genera of the Reoviridaefamily. The ARVs are the cause of some important diseases inpoultry such as reovirus-induced arthritis, tenosynovitis,chronic respiratory disease, and mal-absorption syndrome.OBJECTIVES: In this study, the presence of ARVs in the Iranianbreeder flocks was investigated through reverse transcriptionpolymerasechain reaction (RT-PCR) and restriction enzymefragment length polymorphism (RFLP). METHODS: A total of800 fecal swab samples were initially collected from breederflocks (older than 45 weeks of age). They were then sent to thelaboratory in containers with PBS, and after that they werepooled and finally to 120 samples were obtained. The total RNAextracted from the pooled fecal samples were used to amplify theselected parts of the S1 (1023 bp) and S4 (437 bp) genes from theARV field isolates using RT-PCR. The positive RT-PCRamplified products were further analyzed by RFLP using fiverestriction enzymes. RESULTS: Based on the findings, 5 sampleswere positive with the S1 primer and 6 samples were with the S4one. The patterns observed after the digestion of PCR productsrevealed that the isolates of this study were identical to both theS1133 vaccine and standard strains. CONCLUSIONS: Thefindings suggested that the RT-PCR/RFLP analysis might beconsidered as a simple and rapid approach for the differentiationof ARVisolates. This study was the first molecular detection ofthe ARVs presence in the Iranian breeder flocks using the RTPCRamplification of the S1 and S4 genes and RFLP analysis.