Health Literacy refers to individual competences to access, understand, appraise and apply health information and services to make appropriate health decisions. Studies show that health literacy has crucial influence on one’s health status. For Thailand, there is no a standard tool to assess health literacy at the regional and national level. The present study aimed to develop and validate a Thai health literacy questionnaire for Thai people aged 15 years and above for assessing health literacy at the regional and national level. A cross sectional design was employed to collect data with a developed questionnaire in six provinces. The provinces were Chiang Mai, Nakornsawan, Nakornratchasima, Bangkok, Chonburi and Nakornsrithammarat. The questionnaire was drafted based on Nutbeam’s and European Health Literacy survey framework. It was pre-tested for plain language with experts and 35 general people. For the validation, the questionnaire was administered through face to face interviews. Focus groups were conducted for qualitative analysis. The internal consistency reliability was confirmed by the Cronbach’s alpha. The construct validity was confirmed by the confirmatory factor analysis. Correlations between health literacy scores and health outcomes were also explored. There were 722 people participating in the study. The Cronbach’s alpha was .88 for access, .86 for understanding, .90 for asking and .79 for deciding. All the Cronbach’s alphas are above .70 which indicates a satisfactory internal consistency reliability for the whole questionnaire. The confirmatory factor analysis also shows a good construct validity. Health literacy scores also correlated significantly with health outcomes which included having a chronic disease and metabolic syndrome. Those health outcomes were more prevalent among people with lower health literacy. The Thai Health Literacy Questionnaire (THL-Q) is thus appropriate to assess health literacy at the regional and national level.