Rotavirus genotypes are species specific. However, interspecies transmission is reported to result in the emergence of new genotypes. A cross-sectional study of 242 households with 281 cattle, 418 goats, 438 pigs, and 258 humans in Uganda was undertaken between 2013 and 2014. The study aimed to determine the prevalence and genotypes of rotaviruses across co-habiting host species, as well as potential cross-species transmission. Rotavirus infection in humans and animals was determined using NSP3 targeted RT-PCR and ProSpecT Rotavirus ELISA tests, respectively. Genotyping of rotavirus-positive samples was by G- and P-genotype specific primers in nested RT-PCR assays while genotyping of VP4 and VP7 proteins for the non-typeable human positive sample was done by Sanger sequencing. Mixed effect logistic regression was used to determine the factors associated with rotavirus infection in animals. The prevalence of rotavirus was 4.1% (95% CI: 3.0–5.5%) among the domestic animals and 0.8% (95% CI: 0.4–1.5%) in humans. The genotypes in human samples were G9P and P. In animals, six G-genotypes, G3(2.5%), G8(10%), G9(10%), G11(26.8%), G10(35%), and G12(42.5%), and nine P-genotypes, P(2.4%), P(4.9%), P(7.3%), P(14.6%), P(7.3%), P(9.8%), P(9.8%), P(12.2%), and P(17.1%), were identified. Animals aged 2 to 18 months were less likely to have rotavirus infection in comparison with animals below 2 months of age. No inter-host species transmission was identified.