The aim of the present study was using RT-PCR for the diagnosis of avian infectious bronchitis virus and Massachusetts serotype in tissue samples. Optimization of a molecular diagnostic method for the detection of avian bronchitis virus and identification of Massachusetts serotype was investigated. In order to detect infectious bronchitis virus (IBV) in tissue samples, an RT-PCR was optimized. Specific primers from conserved region of all known IBV serotypes were used in the first PCR assay. Specific primers for the identification of Massachusetts serotype were selected from S-1 gene of the virus in the second PCR reaction. The S-1 gene is a hypervariable region among IBV serotypes; therefore, the amplification of this region is important for serotype identification. Viral RNA was extracted from vaccine and tissue samples from vaccinated and clinical tissue samples of suspected birds. After construction of cDNA, two PCR assays were performed. In the first RT-PCR, detection of virus in test samples was investigated and in the second PCR, Massachusetts serotype was identified. To identify the specificity of the test, PCR amplicons were sequenced. In the first reaction, the IBV was detected in the samples and a 600 bp fragment was amplified. In the second PCR, a 355 bp fragment was amplified from S-1 gene, which confirmed the detection of Massachusetts serotype. In clinical samples, the IBV was also detected. The specificity of the test was confirmed by sequencing of PCR products. Sequence data were submitted to the GenBank which could be accessible by AY954694 number. The RT-PCR is a specific assay for the detection of IBV. Detection of IBV and identification of genetic differences among IBV subtypes in Massachusetts serotype would be possible by sequencing of amplified products.