Occupational stress can affect specialty nurses' quality of work, especially for those working in care units. This study, therefore, investigated role stress and its related factors among specialty nurses working in tertiary general hospitals. This cross-sectional descriptive study used convenience sampling to recruit 795 Chinese specialty nurses in 11 tertiary general hospitals (from February to March 2023). A questionnaire survey was conducted using the Basic Information Questionnaire and the Role Stress Scale. Multiple linear regression analyses were performed on the survey data to explore the factors affecting role stress. The total role stress score of specialty nurses in tertiary general hospitals was 52.05 ± 19.98. The highest mean item score was quantitative overload, followed by qualitative overload, role conflict, and role ambiguity, which had the lowest score. Multiple linear regression analysis revealed that gender (β = -0.085, p < 0.05), educational background (β = 0.077, p < 0.05), and work experience (β = -0.104, p < 0.05) were the main factors influencing role stress among specialty nurses. Specialty nurses in tertiary general hospitals had higher levels of role stress than general nurses. Their role stress was primarily reflected in role overload, followed by role conflict and ambiguity. The factors affecting specialty nurses' role stress included gender, work experience, and educational background. Nursing managers should monitor the role stress experienced by specialty nurses in tertiary general hospitals. Providing psychological support for male specialty nurses, performance rewards and learning opportunities for highly educated specialty nurses, and continuous training for inexperienced specialty nurses are essential measures to relieve role stress.