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Background: There is little information regarding the hepatitis B virus (HBV), vaccination status, and hepatitis B exposure in Italian women's jails. We aimed to describe the HBV exposure and HBs antibody (anti-HBs) protection levels in female prisoners. Material and methods: A retrospective multicentric study was performed in Italian prisons from 2021 to 2023. Univariate and multivariate analyses were conducted to identify risk factors for HBc antibody (anti-HBc) seropositivity and non-protective anti-HBs titer. Results: We included 156 patients. The median age was 41.0 (IQR 34.0–48.0). Of the studied subjects, 31 (19.9%) had anti-HBc positive titer. Two women were HBsAg positive. In the multivariate analysis, older age [OR 1.06 (CI 1.01–1.11), p = 0.011], North-Eastern European [OR 11.67 (3.29–41.30), p < 0.001] and African origin [OR 6.92 (CI 1.51–31.60), p = 0.013], and drug use [OR 6.55 (CI 1.96–21.9), p = 0.002] were risk factors for HBV exposure. Thirty-seven (32%) women had no history of HBV vaccination. Forty-four (38%) had an anti-HBs non-protective titer. In the multivariate analysis, North-Eastern European origin [OR 4.55 (CI 1.19–17.50), p = 0.027] was associated with unprotective anti-HBs titer. Conclusion: Our results show both the low prevalence of HBV and protection in female prisoners. Age, North-Eastern European and African origin, and drug use have a role in exposure risk to HBV. [ABSTRACT FROM AUTHOR]