Abstract Background The assessment of TLR agonists as promising candidate adjuvants for the induction of effective Th1 immunity continues to rely on the use of inbred mouse. However, the genetic variation among commonly used inbred mice may influence the efficacy of candidate adjuvants and bias the study conclusion. Indeed, we have found significant differences in the recruitment, phenotypes and cytokines production of DCs and NK cells and the generation of effector T cell in lymphoid tissues of two genetically non-identical mouse strains immunized with a protein based vaccine formulated with TLR agonists. Objective In this study, we further evaluated the nature and amplitude of humoral immune responses of B6 and BALB/c strains immunized with protein antigen ovalbumin plus Alum, TLR3, TLR4, TLR7/8, or TLR9 agonists. Results Our data suggest that compared to the Alum, all other adjuvants induced more effectively the recruitment of B cells and production of OVA-specific antibodies in both strains. We also observed significant differences between two strains. BALB/c strain recruited less B cells but produced significantly higher amounts of IgG2a in response to any of the TLR agonists. In addition, both strains produced similar amounts of IgG1 and IgG2b in response to TLR agonist except for MPLA which induced significantly more of IgG2b production in B6 strain. Conclusion Thus, our data suggests that genetic background should be taken into consideration when evaluating and comparing the activities of adjuvants for use in new prophylactic and therapeutic vaccines. NIH-NIAID 1R03AI103750-01A1