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Background: Despite increased access to HIV pre-exposure prophylaxis (PrEP) in Canada, familiarity and experience among primary care providers (PCPs)—including family doctors and those working with key populations—remains limited. To understand the barriers and facilitators of PrEP familiarity and experience, we conducted a situational analysis in PCPs in sub-urban and rural Ontario. Methods: We surveyed a non-probabilistic sample of PCPs using an online questionnaire, designed with the Consolidated Framework for Implementation Research (CFIR). Poisson regressions with robust variance were used to assess the relationship between CFIR domains, sociodemographic, and practice characteristics on both PrEP familiarity and experience. Results: A total of 54 PCPs participated (6% response rate), comprising 80% physicians and 20% nurses. Nearly 30% of the sample worked with key populations, including sexual health clinics and community care centers, 18% of respondents reported high familiarity with PrEP, and 44% reported PrEP experience (referred, started a conversation, or prescribed). PrEP familiarity and experience were associated with working in an organization serving key populations, working with gender minorities, and having colleagues providing PrEP. Providers with a positive perception of PrEP and its necessity for populations at risk were more likely to have PrEP-related experience. Higher familiarity and experience were reported by PCPs with specific clinical skills related to PrEP, and with the perception that PrEP was compatible with their practice as primary provider. Conclusions: Our findings suggest that organizational support, and additional training and education would facilitate PrEP provision by PCPs in suburban/rural Ontario.