Smoking is the leading cause of premature mortality and morbidity. The aim of this study was to provide the first national description of organizational capacity and involvement in tobacco control (TC) measures outlined by the WHO Framework Convention on Tobacco Control (FCTC) within the Czech Republic. Data were collected in a national cross-sectional survey of all 14 organizations engaged in TC activities within the Czech Republic. Organizational capacity (defined as skills, supports, partnerships, resources, and leadership) to implement TC activities, and level of involvement in key FCTC measures were assessed and compared across organizations. Despite the high economic costs of tobacco use, few organizations were involved in TC activities. 50% of all organizations involved in TC activities were non-government or non-profit organizations. Less than one third of organizations reported having a sufficient number of staff or adequate funding to work effectively. Skills for chronic disease prevention (CDP) practice including assessment, identifying relevant practices, developing and implementing initiatives were rated more favourably than skills to evaluate these activities. Level of involvement was ranked highest for activities that focused on creation of smoke-free environments and lowest for activities that focused on raising taxes and sales to minors. Organizations tended to be more involved in individual, rather than population-level prevention strategies. Inadequate funding, insufficient number of staff dedicated to working on TC, and lack of political will were major barriers. This paper provides the first national description of organizational capacity and level of involvement in FCTC measures within the Czech Republic.