AbstractRotavirus causes significant morbidity and mortality among children worldwide. Stool samples from a previous hospital-based surveillance study to detect diarrhea etiology at the National Pediatric Hospital in Phnom Penh, Cambodia, by Meng and others in 2011 were tested for rotavirus by real-time reverse transcription polymerase chain reaction (PCR) targeting vp6 gene and characterized for G- and P-genotypes of positive samples based on vp7 and vp4 genes, respectively. Rotavirus was detected in 159/531 (30%) of children with diarrhea and none was detected in 287 nondiarrhea controls. All but three of the rotavirus-positive cases were children under the age of 2. The most common genotypes characterized by PCR and sequencing were G1P[8] (69%), G9P[8] (11%), and G2P[4] (11%). Genotype G9 was detected at a relatively high percentage that is consistent with the global trend and found to be associated with hospitalization. Data on disease burden and genotypic distribution are required information for the planning of rotavirus vaccine implementation in Cambodia.