BACKGROUND:: Rotavirus is the most common cause of severe diarrhea among children <5 years of age worldwide. To determine the prevalence of severe rotavirus infection in children admitted with acute diarrhea attending Mulago National Referral Hospital in Uganda, active sentinel surveillance was conducted from July 2006 to December 2012. METHODS:: A total of 6387 children with acute diarrhea were enrolled into the public health surveillance system and of these, 5627 had stool samples collected and tested for rotavirus antigens by enzyme immunoassay ProSpecT Rotavirus kit. RESULTS:: Rotavirus was detected in 1844 (32.8%) of 5627 children with acute diarrhea that had stool specimens collected, and 93% of positive cases of rotavirus gastroenteritis were between 3 and 23 months of age, with highest prevalence in children 6-11 months of age. Rotavirus infections occurred throughout the year. During the surveillance period (2006-2012), a total of 354 positive stool samples were subjected to reverse transcription polymerase chain reaction and genotyping assays. The most common genotypes detected were G1P (16.1%) and G9P (15.3%), followed by G2P (7.6%), G9P (7.1%), G8P (6.5%) and G12P(5.6%). Mixed G or P types (17.9%) and partially typed either G or P types (10.7%) were common. CONCLUSION:: Uganda would benefit by introducing rotavirus vaccine and hence reduce the hospitalization burden of managing acute diarrhea cases.