Abstractobjectives To assess the burden of rotavirus disease in Guatemala, in view of the recent introduction of a national rotavirus vaccination programme.methods We examined data from an active, facility-based surveillance system in Santa Rosa, Guatemala, from October 2007 through September 2009 among children <5 years of age presenting to the hospital or ambulatory clinics with diarrhoea ( ‡3 loose stools in 24 h during the last 7 days). Demographic and epidemiological data were collected, and specimens were tested for rotavirus via enzyme immunoassay. Genotyping was performed via reverse transcriptase polymerase chain reaction.results We enrolled 347 hospitalized patients <5 years of age with diarrhoea and 1215 from ambulatory clinics. Specimens from 275 (79%) hospitalized children and 662 (54%) from ambulatory visits were tested for rotavirus. Rotavirus accounted for 32% of hospitalizations and 9% of ambulatory visits for diarrhoea, resulting in adjusted annual rates of 36 hospitalizations and 372 ambulatory visits per 10 000 children. Ninety-one per cent of hospitalizations and 81% of ambulatory visits for rotavirus diarrhoea occurred in children <2 years. G1P8 represented 71% and 95% of rotavirus genotypes for 2007-2008 and 2008-2009 rotavirus seasons, respectively.conclusions Rotavirus is a major cause of diarrhoea in children <5 years of age in Santa Rosa, Guatemala, highlighting the potential health benefits of vaccination and the need for continued surveillance to assess impact and effectiveness of the rotavirus vaccination programme in Guatemala.