Rapid detection and differentiation of infectious bronchitis virus (IBV) involved in the disease outbreak is very important for controlling disease and developing new vaccines. In the present study, three regions of the genome of IBV vaccine and field isolates including S1 gene, gene 3 and nucleocapsid (N) gene along with 3' untranslated region (3' UTR) were amplified and subjected to restriction fragment length polymorphism (RFLP) using three different endonucleases. Amplicons from S1 gene and N-3’UTR generated four RFLP patterns, grouping IBV strains into four similar groups, while amplicons of gene 3 generated three RFLP patterns classifying examined IBVs in different groups from those of S1 and N-3' UTR. 4/91 strain and MNS-7862-1field isolate both belong to 793/B serotype were differentiated from each other based on gene 3, N-3’UTR and S1gene. IBVs belonged to different serotypes showed different RFLP patterns based on RFLP patterns of all three regions. S1 gene and N-3’UTR RFLP analysis differentiated IB88, MNS-7862-1 and 4/91 from each other. This is the first report on the molecular analysis of the gene 3 for IBV strain differentiation. Our results revealed that RFLP analysis of N-3’UTR and S1 gene had the higher discriminatory power than gene 3. None of the RFLP patterns of different regions differentiated 4/91 vaccine strain from its field isolate.