Objective: HIV testing is important for HIV prevention and control. Nevertheless, approximately 35% of HIV infections in Ontario remain undiagnosed. We examined patterns of HIV testing among physicians to determine provider-related factors associated with HIV testing in Ontario. Methods: Providers identified from HIV test requisitions submitted to the Ontario Public Health Laboratory in 2006 were linked to a database of Ontario physicians using probabilistic matching. We examined HIV testing frequency by demographic characteristics and physician specialty. In multivariate logistic regression, we assessed factors associated with high testing frequency (20+ tests). Results: 12,477 physicians (59.3% of Ontario physicians) prescribed at least one HIV test in 2006; the proportion was highest in Central East/other (72.4%) and Northern (69.4%) regions compared to others (53.7-58.7%), and highest among the most recent graduates (68.4% versus 59.0% among earlier graduates). A substantial proportion of physicians in family medicine/general practice (83.6%), obstetrics/gynecology (82.1%) and internal medicine (47.7%) prescribed HIV testing. Overall, most physicians (67.5%) prescribed fewer than 20 tests. High testing frequency was significantly associated with practice in Toronto (AOR 2.95), Central East/other (AOR 2.02), or Ottawa region (AOR 2.28), and specialty in family medicine/general practice (AOR 11.47), obstetrics/gynecology (AOR 6.31) or internal medicine (AOR 1.37). Physician sex and graduation country were not associated with high testing in multivariate regression. Of 361,609 tests, 1,048 (0.29%) were HIV-positive; 436 physicians (3.5%) had at least one HIV-positive result. Conclusion: HIV testing among Ontario physicians varied substantially by health region, graduation year and specialty. These factors should be considered when delivering continuing medical education on HIV testing.