Background: Noroviruses are one of the major viral pathogens responsible for gastroenteritis. Outbreaks of diarrhea due to Norovirus have been reported frequently. This study is performed to determine the prevalence of Norovirus in fecal specimens of children with gastroenteritis. Many viruses can cause gastroenteritis, including Rotaviruses; Adenoviruses types 40 and 41; Sapoviruses; and Noroviruses. Current techniques used for detection of Noroviruses in stool samples include multi-step viral RNA extraction and purification followed by reverse transcriptase-polymerase chain reaction (Rt-PCR). Objectives: The purpose of this study is to detect Norovirus in stool samples by Rt-PCR in 5 different centers in Iran. Patients and Methods: In this study, 2,170 stool samples were collected from children less than five years old from five different cities, all of whom had acute gastroenteritis. Detection of Noroviruses was performed through Rt-PCR. The mean age of the studied population was 48 months. Fecal specimens were collected within 24 hours of admission. The specimens were frozen, sent to the laboratory, and then stored at -70° C until being tested for Norovirus. Results: Rt-PCR was performed for 2,170 stool samples containing 90 (4.14%) Norovirus positive (0.97% Tehran, 0.64% Tabriz, 0.18% Mashhad, 1.57% Shiraz, 0.78% Bandar Abbas). The RT-PCR was validated with published primers for Norovirus (JV12/JV13). In both retrospective and prospective settings, the Rt-PCR was equally sensitive (95%) and specific (95%) in detecting Norovirus. Conclusions: Noroviruses, which are important human pathogens, may cause epidemic acute viral gastroenteritis which in turn can be easily detected by molecular methods.