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A multicenter study was conducted to estimate the prevalence of pertussis IgG antibodies (anti-PTx) in the Italian population. Serum samples (4154) collected in the years 2019–2020 from subjects aged 6 to 90 years were tested. The anti-PTx IgG levels were determined by ELISA test. The limit of detection was 5 IU/mL (International Units per milliliter); values ≥ 40 IU/mL and ≥100 IU/mL indicate an infection that has occurred in recent years and a recent infection (occurred during the last year), respectively. The mean concentration of anti-PTx IgG antibodies in the tested samples was 13 IU/mL; 1.0% of subjects had a titer ≥ 100 IU/mL, 5.3% a titer between 40 and 100 IU/mL, and 38.9% a titer < 5 IU/mL. The mean antibody concentration was significantly higher in males than in females. The age group 25–39 years had the lowest percentage of negative subjects (36.9%) and the highest prevalence of subjects with antibody titers ≥ 100 IU/mL (1.3%). In the age group ≥ 65 years, the prevalence of subjects with titers between 40 and 100 IU/mL (6.7%) and the percentage of negative subjects (44.8%) was higher than in the other age groups. The results highlight the possible role of adolescents and adults in the transmission of B. pertussis.