Human rotaviruses (HRV) are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Therefore, the early diagnosis is essential for effective patient management and infection control. We have developed a rapid, simple technique for the diagnosis of rotavirus based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in vertical agarose gels and developed by silver staining (AGE-SS). This method also has the ability to detect differences in the electrophoretic mobility of RNA bands among group C rotaviruses, reovirus, and group A rotaviruses. The results indicate that this assay is as sensitive and specific as the polyacrylamide gel electrophoresis and silver stain method (PAGE/SS) and it could be applied on large scale for the screening of stool suspected of rotaviral diarrhea. This assay does not need sophisticated equipment and the cost per sample is minimal compared with other available assays.