Background: In order to end the HIV epidemic by 2030, combination HIV prevention including pre-exposure prophylaxis (PrEP) should be widely available, especially for the most vulnerable populations. In Latin America and the Caribbean (LAC), only 14 out of 46 countries have access to PrEP. In Brazil and Mexico, PrEP has been provided at no cost through the Public Health System since 2017 and 2021, respectively. Thus, HIV physicians’ perspectives about PrEP and other prevention strategies may differ. This study aimed to compare awareness, knowledge, and attitudes related to PrEP and other prevention strategies among HIV physicians from Brazil and Mexico. Methods: Cross-sectional, web-based survey targeting physicians who prescribe antiretrovirals from both countries. Participants answered questions on socio-demographic, medical experience, awareness, knowledge, and attitudes towards PrEP and other HIV prevention strategies. We stratified all variables per country and compared frequencies using Chi-square, Fisher exact, and Wilcoxon-Mann-Whitney tests, as appropriate. Results: From January–October 2020, 481 HIV physicians were included: 339(70.5%) from Brazil, 276(57.4%) male, and median age was 43 years (IQR = 36–53). Awareness of PrEP did not differ between Brazil and Mexico (84.6%), while awareness of other prevention strategies, including post-exposure prophylaxis and new PrEP technologies, was higher in Brazil. More Brazilians perceived U=U as completely accurate compared to Mexicans (74.0% vs. 62.0%, P <.001). Willingness to prescribe PrEP was 74.2%, higher among Brazilians (78.2%, P =.01). Overall, participants had concerns about consistent access to PrEP medication and the risk of antiretroviral resistance in case of acute HIV infection or seroconversion. The main barriers reported were assumptions that users could have low PrEP knowledge (62.0%) or limited capacity for adherence (59.0%). Compared to Brazilians, Mexicans reported more concerns and barriers to PrEP prescription (all; P ≤.05), except for consistent access to PrEP medication and the lack of professionals to prescribe PrEP (both; P ≤.01). Conclusions: Although awareness of PrEP was similar in Brazil and Mexico, differences in knowledge and attitudes may reflect the availability and stage of PrEP implementation in these countries. Strengthening and increasing information on PrEP technologies and other HIV prevention strategies among HIV physicians could improve their comfort to prescribe these strategies and facilitate their scale-up in LAC.