The capsular polysaccharide of the human pathogen Group B Streptococcus is a key virulence factor and vaccine candidate that induces protective antibodies when conjugated to carrier proteins. It consists of long polymeric chains of oligosaccharide repeating units, and each of the ten capsular serotypes described so far presents a unique chemical structure with distinct antigenic properties; therefore, broad protection against this pathogen could be achieved by a combination of ten glycoconjugates. Capsular polysaccharide biosynthesis and assembly follow a polymerase-dependent pathway that is widespread in encapsulated bacteria and is encoded by a polycistronic operon. Here we exploited the sequence similarity between the capsule operons of types V and IX to generate hybrid polysaccharides incorporating epitopes of both serotypes in a single molecule, by co-expressing their specific CpsM, O, I glycosyltransferases in a single isolate. Physicochemical and immunochemical methods confirmed that an engineered strain produced a high molecular weight chimeric polysaccharide, combining antigenic specificities of both type V and IX. By optimizing the copy number of key glycosyltransferase genes, we were able to modulate the ratio between type-specific epitopes. Finally, vaccination with chimeric glycoconjugates significantly decreased the incidence of disease in pups born from immunized mice challenged with either serotype. This study provides proof of concept for a new generation of glycoconjugate vaccines that combine the antigenic specificity of different polysaccharide variants in a single molecule, eliciting a protective immune response against multiple serotype variants.