Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure histories become increasingly complex through original and variant-adapted vaccines and infections with viral variants. Upon exposure to the highly altered Omicron spike glycoprotein, pre-immunized individuals predominantly mount recall responses of Wuhan-Hu-1 (wild-type)-imprinted memory B (BMEM) cells mostly targeting conserved non-neutralizing epitopes, leading to diminished Omicron neutralization. We investigated the impact of imprinting in individuals double/triple vaccinated with a wild-type-strain-based mRNA vaccine who, thereafter, had two consecutive exposures to Omicron BA.1 spike (breakthrough infection followed by BA.1-adapted vaccine). We found that depletion of conserved epitope-recognizing antibodies using a wild-type spike bait results in strongly diminished BA.1 neutralization. Furthermore, spike-specific BMEM cells recognizing conserved epitopes are much more prevalent than BA.1-specific BMEM cells. Our observations suggest that imprinted BMEM cell recall responses limit the induction of strain-specific responses even after two consecutive BA.1 spike exposures. Vaccine adaptation strategies need to consider that prior SARS-CoV-2 infections and vaccinations may cause persistent immune imprinting. Display omitted •Two exposures to Omicron BA.1 spike boost BA.1 neutralization in pre-vaccinated individuals•BA.1 neutralization is largely mediated by antibodies cross-recognizing wild-type SARS-CoV-2•BA.1-specific neutralizing antibodies are detectable only in some individuals•Memory B cells recognizing conserved epitopes predominate over BA.1-specific B cells Muik et al. show that, in individuals pre-immunized with SARS-CoV-2 ancestral strain mRNA vaccines, cross-reactive humoral immunity targeting conserved epitopes remains dominant after two exposures to Omicron BA.1 spike. Their observations indicate that persistent immune imprinting may limit strain-specific naive B cell priming even after repeated variant spike exposure.