Background: Human caliciviruses (HuCV) are emerging enteric pathogens that are a common cause of diarrhea in humans worldwide. The prevalence of HuCV responsible for acute gastroenteritis in children under 5 years old was determined using Reverse transcription-Polymerase Chain Reaction (Rt-PCR) on purified viral nucleic acid from fecal samples. Objectives: The aim of this study was to detect Calicivirus in stool samples by Rt-PCR in 5 different centers in Iran. Methods: In this study, 3260 stool samples were collected from children less than 5 years old with acute gastroenteritis from five different cities. Calicivirus detection was performed through Rt-PCR. Fecal speciments were collected within 24 hours of admission. The speciments were frozen, sent to the laboratory, and then stored at -80 0C until being tested for Calicivirus. Results: Rt-PCR was performed for 3260 stool samples containing 53 (1.62%) HuCV positive. The Rt-PCR was validated with published primers for HuCV (P289/P290). 84.2% of Calicivirus-caused gastroenteritis was found among children under 2 years old, which was statistically significant (P value < 0.05). Conclusion: HuCVs, one of the most important causes of acute gastroenteritis in children under 5 years old, can be detected by Rt-PCR.