Abstract Chronic infectious diseases caused by viral pathogens such as HIV and Hepatitis C constitute a major global health challenge. Chronic viral infections result in poor humoral immune responses, characterized by the delayed generation of neutralizing antibodies and dysregulation of memory B cell formation. Little is known regarding the mechanisms driving dysfunctional B cell memory during chronic viral infection. We used an in vivo mouse model using acute and chronic strains of LCMV, in conjunction with a B cell tetramer specific for the LCMV nucleoprotein, to understand changes that occur in antigen-specific memory B cell formation in acute vs. chronic viral infection. We performed phenotypic analyses of genetically modified mouse strains, BrdU-labelling studies, antiviral treatment experiments and single-cell RNA-sequencing to examine memory B cell formation, phenotype and function in response to acute vs. chronic LCMV infection. Single-cell RNA-sequencing identified key genes downregulated in chronic memory B cells associated with complement-mediated immune functions. We found that T-bet and CXCR3 modulate multiple aspects of the humoral immune response in chronic viral infection, including GC B cell localisation, memory B cell phenotype, and immune complex expansion. Our data provides novel insight into the cellular and molecular mechanisms driving altered humoral immune responses in chronic viral infection.