Rotavirus is the leading cause of severe diarrhea in children under five years worldwide. It is ranked as a priority for vaccine. In Burundi, vaccine against rotavirus was implemented in 2013. The impact of recent rotavirus vaccination on morbidities in Burundi is not well established. Moreover, no study has been carried out to document the genetic diversity of rotavirus strains circulating in Burundi. This cross-sectional health facilitybased study aimed at determining the prevalence and molecular characteristics of rotavirus infections among children under five years of age in Mutaho Health District and the Municipality of Bujumbura, in Burundi. Stool specimens were collected from children presenting with acute diarrhea. These specimens were tested for rotavirus antigen using Diagnostar ® rapid test kit. Positive stool samples were confirmed at the Kenya Medical Research Institute (KEMRI) by ELISA. Positive confirmed samples underwent RT-PCR, G and P genotyping by multiplex semi-nested PCR using a cocktail of type specific primers or by sequencing. A total of 646 participants were enrolled in this study. The overall prevalence of rotavirus was 6.2% (40/646) with 4.0% (16/400) in Mutaho health district and 9.7% (24/246) in the Municipality of Bujumbura. Rotavirus detection rate tended to increase as the level of precipitation went down, showing a significant negative association between the two variables. (OR = 15.2; P = 0.0001). In addition, rotavirus detection rate was higher in Bujumbura Municipality than in Mutaho health district (OR = 2.6; P = 0.005). Two G genotypes were identified, G1 the predominating G genotype accounted for 53.8% (14/26) followed by G12 (46.2%, 12/26). The prevalence of the genotype G1 of Group A rotavirus was significantly higher in Bujumbura Municipality than in Mutaho health district while G12 predominated in Mutaho health district (OR = 7.33; P = 0.026). Rotavirus strains from pigs might have contributed to the high prevalence of human G12 rotavirus in that area. Three different P types were identified P[8] the most common, followed by P[6] and P[4]. The most common G/P combination genotype was G1P[8] which accounted for 45.5% of all rotavirus genotypes identified, followed by G12 P [8] (41.0%), G1P [6] (4.5%), G12 P [6] (4.5%) and G12 P [4] (4.5%). The emergence of G12 rotavirus strains which share neither G nor P genotypes with currently used rotavirus vaccines raises public health concerns as they have the potential to challenge their efficacy. Therefore, we recommend to initiate and maintain a continuous rotavirus strain surveillance in Burundi so as to monitor trends in the occurrence of these prevailing and potentially emerging new strains.