Background: HRV is the causative agent of severe gastroenteritis in children and responsible for two million hospitalizations and more than a half-million deaths annually. Sequence characteristics of the gene segments encoding the VP7 and VP4 proteins are used for the genotype classification of rotavirus. A wide variety of molecular methods are available, mainly based on reverse transcription PCR for rapid, specific and sensitive genotyping of rotaviruses. This study describes an alternative Real-Time PCR assay for genotyping of rotavirus. Methods: The samples of stools studied in this research have been collected from patients referred to Children's Medical Centers, Tehran, Iran. Rotavirus detection and genotyping were performed using the RT-PCR and seminested RT-PCR, respectively. Samples were then genotyped with a new Real-Time PCR. Results: The Real-Time PCR was able to genotype all positive samples with a mean Ct of 28. 2. Besides, a concordance rate of 100% was detected between Real-Time PCR and semi-nested RT-PCR. Conclusion: In this study, the genotyping of rotavirus with Real-Time PCR showed that this method can provide several favorable features, including high sensitivity and specificity, and a wide dynamic range for rotavirus genotyping.