Women have been historically underrepresented in both craft and management positions in the U.S. rail industry. Identified as one of the industry’s top workforce challenges, the present study provides a more detailed view of gender diversity in rail transportation, and how factors such as gender and management status interact to affect perceptions of safety culture in a sample of U.S. short line and regional railroads (Nrailroads = 23, Nrailroaders = 1,239). The results suggested that women tended to be older than men, have more education, and were more likely to be employed at larger organizations. Despite being employed in the railroad industry for fewer years than men, women in our sample were also more likely to be managers. With regard to safety culture, managers had more positive perceptions than craft employees, and female craft employees had more positive perceptions than male craft employees. Implications for the industry and avenues for future research and implementation are discussed.