BACKGROUND Rotavirus infections frequently cause acute gastroenteritis in humans and are the most important cause of severe dehydrating diarrhea in young children in both developed and developing countries. METHODS This was a prospective cross-sectional, hospital-based study on 300 children ≤ 5 years with acute watery diarrhea who attended Bugando Medical Centre (BMC) and Nyamagana District hospital between May and November 2009. Stool specimens were tested for rotavirus infection using latex agglutination test. Data were cleaned and analyzed using SPSS 11.0. RESULTS Of 300 children with acute watery diarrhea, 136 (45.3%) were female and the mean age was 12.63 months (SD = 10.4). Sixty-two (20.7%) children were found to have rotavirus infection. Of children with severe malnutrition three (37.5%) were infected with rotavirus. Fifty-two (84%) of children with rotavirus infection were below two years of age. Severe dehydration was present in 48 (16%) children of whom 12 (25%) were infected with rotavirus compared to 18 (16.6%) of 109 children with no dehydration. Living next door to a child with diarrhea was highly associated with rotavirus infection (43% versus 19%; p = 0.036). The mean hospital stay among children with rotavirus infection was 3.66 days versus 2.5 days for those without rotavirus (p = 0.005). CONCLUSIONS Rotavirus infection is prevalent in Mwanza region and contributes to prolonged hospital stay. Proper education on hygiene to control diarrheal diseases among children should be emphasized. Extensive studies to determine the serotypes of rotavirus are warranted in the region before rotavirus vaccine is introduced.