Abstract Background Maternal breastmilk is a source of pre- and pro-biotics that impact neonatal gut microbiota colonization. Because oral rotavirus vaccines (ORVs) are administered at a time when infants are often breastfed, breastmilk microbiota composition may have a direct or indirect influence on vaccine take and immunogenicity. Methods Using standardized methods across sites, we compared breastmilk microbiota composition in relation to geographic location and ORV response in cohorts prospectively followed from birth to 18 weeks of age in India (n = 307), Malawi (n = 119), and the United Kingdom (UK n = 60). Results Breastmilk microbiota diversity was higher in India and Malawi than the UK across 3 longitudinal samples spanning weeks of life 1 to 13. Dominant taxa such as Streptococcus and Staphylococcus were consistent across cohorts; however, significant geographic differences were observed in the prevalence and abundance of common and rare genera throughout follow up. No consistent associations were identified between breastmilk microbiota composition and ORV outcomes including seroconversion, vaccine shedding after dose 1, and postvaccination rotavirus-specific immunoglobulin A level. Conclusions Our findings suggest that breastmilk microbiota composition may not be a key factor in shaping trends in ORV response within or between countries. Breastmilk microbiota composition differed significantly between cohorts in India, Malawi, and the UK. No consistent associations were identified between breastmilk microbiota composition and infant oral rotavirus vaccine response.