Context: Hamstring strain injuries (HSIs) are the most frequently sustained injury in Major League Baseball (MLB). However, the beliefs and practices of MLB practitioners regarding HSI risk factors and prevention strategies in baseball athletes, have not been documented. Objective: To document the current beliefs and practices of MLB practitioners in relation to HSI prevention. Design: cross-sectional study. Setting: Major League Baseball via an online survey. Participants: Athletic trainers, physical therapists and strength and conditioning coaches employed in MLB during the 2021 season. Data collection and analysis: An online survey was conducted with participants completing the survey once. Questions pertained to risk factor identification, the use and perceived effectiveness of preventative strategies, and barriers to implementation. Descriptive statistics were calculated for each question. Results: 91 responses were received featuring respondents from 28 of 30 MLB organizations. The perceived most important intrinsic risk factor for first-time HSI was tolerance to high-speed running and previous HSI for recurrent injury. The perceived most important extrinsic risk factor for both first-time and recurrent HSI was internal communication between staff. The perceived most effective prevention strategies were managing overall workload, exposure to high-speed running, and periodization. The most used prevention strategies were core/lumbopelvic strengthening, resistance training and workload management. Approximately half (53%) of respondents reported barriers to effective implementation of HSI prevention strategies, including player and coach buy-in, compliance, training time constraints, and in-season scheduling/reduced recovery time. Conclusions: This was the first survey to investigate MLB practitioner beliefs and practices regarding HSI prevention. Responses from practitioners regarding their beliefs about risk factors and appropriate prevention strategies were varied, and discrepancies existed between the perceived most effective strategies and those most frequently employed.