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Rotavirus vaccination has reduced mortality and hospital admissions due to rotavirus diarrhoea, but its effect on rotavirus infections and the impact of rotavirus genotypes are still unclear. Real-time PCR was used to detect rotavirus and other pathogens in faeces samples from children below five years of age with acute diarrhoea, collected before (n = 827) and after (n = 807, 92% vaccinated) the introduction of vaccination in Rwanda in 2012. Rotavirus was genotyped by targeting VP7 to identify G1, G2, G3, G4, G9 and G12 and VP4 to identify P[4], P[6] and P[8]. In vaccinated children, rotavirus infections were rarer (34% vs. 47%) below 12 months of age, severe dehydration was less frequent, and rotavirus was more often found as a co-infecting agent. (79% vs 67%, p = 0.004). Norovirus genogroup II, astrovirus, and sapovirus were significantly more often detected in vaccinated children. The predominant rotavirus genotypes were G2P[4] and G12P[6] in 2009-2010 (50% and 12%), G9P[8] and G1P[8] in 2011-2012 (51% and 22%), and G12P[8] in 2014-2015 (63%). Rotavirus vaccination in Rwanda has reduced the severity of rotavirus gastroenteritis and rotavirus infection frequency during the first year of life. Rotavirus infections were frequent in vaccinated children with diarrhoea, often as co-pathogen. Rotavirus genotype changes might be unrelated to vaccination because shifts were observed also before its introduction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kabayiza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)