This study aimed to describe the epidemiology of rotavirus infections in Mozambique before vaccine introduction. Between February 2012 and September 2013, stool specimens, demographic and clinical data were collected from 384 children <5 years old hospitalized with acute diarrhea in Mavalane General Hospital and Manhiça District Hospital, southern Mozambique. The samples were tested for rotavirus A using enzyme-linked immunosorbent assay. The overall prevalence of rotavirus infection was 42.4% [95% confidence interval (95CI): 37.4-47.6%], and was similar in Manhiça (44.3%; 95CI: 36.2-52.7%) and Mavalane (41.3%; 95CI: 34.9-47.9%). The highest prevalence of rotavirus infection was observed in children between 6 and 11 months old. It was also observed that 162 (43.7%) of the children were underweight (weight-for-age z-score < -2), of which 61 were infected by rotavirus.