Tobacco use is disproportionately elevated among patients with substance use disorders relative to the general U.S. population. Tobacco interventions are lacking within substance use treatment centers (SUTCs) due to lack of knowledge and training. This study examined knowledge gain and the organizational factors that might moderate knowledge gains following tobacco education training provided to employees (N = 580) within 15 SUTCs that were participating in a tobacco-free workplace program. The number of total annual patient visits, unique annual patient visits, number of full-time employees, and organizational readiness for implementing change (ORIC) as assessed prior to implementation were examined as potential moderators. Results demonstrated significant knowledge gain (p < 0.001) after training overall; individually, 13 SUTCs had significant knowledge gain (p’s < 0.014). SUTCs with fewer total annual patient visits and fewer full-time employees showed greater knowledge gains. The ORIC total score and all but one of its subscales (Resource Availability) moderated knowledge gain. SUTCs with greater initial Change Efficacy (p = 0.029), Valence (p = 0.027), and Commitment (p < 0.001) had greater knowledge gain than SUTCs with lower scores on these constructs; SUTCs with greater Task Knowledge (p < 0.001) regarding requirements for change exhibited less knowledge gain. Understanding the organizational-level factors impacting training effectiveness can inform efforts in organizational change and tobacco control program implementation.