Context: Athletic training is at the forefront of the prevention of injury and illness. As the push for population-level approaches continues, including the expansion of standards related to professional preparation, there is a need to examine the knowledge and practice application of public health topics of athletic trainers (ATs), as they may serve as role models or preceptors. Objective: To assess ATs' knowledge and practice application of public health topics. Design and Setting: Online survey with knowledge assessment. Participants: Four hundred eighty-seven ATs (age = 35.8 ± 11.1 years, years credentialed = 12.8 ± 10.6) voluntarily participated. Intervention(s): The instrument included a demographic section, a pre and post perceived-knowledge assessment, a practice-behavior frequency matrix, and the Public Health Assessment Tool (PHAT) developed by the authors and Delphi panelists. Main Outcome Measure(s): Measures of central tendency were calculated for the practice-behavior frequency matrix. Perceived knowledge was compared pre- and post-PHAT. We calculated a total PHAT score to measure actual knowledge, and compared perceived and actual knowledge to determine if a knowledge gap existed. Results: A significant difference (P > 0.001) was identified for perceived knowledge before and after the PHAT. On the PHAT, participants scored 12.14 ± 2.21 out of 19. The 3 most commonly missed questions directly related to the 3 least practiced topics: social determinants of health, assessing environmental health factors, and assessing health-related quality of life. When exploring the relationship between perceived and actual knowledge, we identified a weak, significant relationship between post-PHAT perceived-knowledge mean and actual-knowledge assessment scores (r = 0.105, P = 0.022). Conclusions: A knowledge gap exists concerning public health topics in ATs. A lack of knowledge related to public health topics identifies the need for ATs to be mindful of the intersection of daily clinical practice and population health with a future emphasis on deploying community-based health promotion.