This study provides a checklist of the phanerogams and pteridophytes of the Tocantins river middle basin, in northern Goiás state and southern Tocantins state, Brazil. Herbarium samples were collected from 2000 to 2009 and this floristic survey recorded 1572 species from 135 families. The most species-rich families were Fabaceae (217), Poaceae (116), Asteraceae (88), Euphorbiaceae (65), Orchidaceae (58) and Malpighiaceae (56). Furthermore, 14 endangered species and 31 rare species were recorded, mainly associated with the campos rupestres in the Veadeiros Plateau region. The flora mainly from the phytophyisiognomies cerrado stricto sensu, campo rupestre (“rocky fields”), mata de galeria (“gallery forest”), mata ciliar (“riverine forest”) and semi-deciduous seasonal forest comprised typical species of the mid-western Cerrado floristic province, such as the most widely known woody plants Anadenanthera colubrina (Vell.) Brenan (popular name angico), Aspidosperma subincanum Mart. ex A. DC. (guatambú), Astronium fraxinifolium Schott ex Spreng. (gonçalo-alves), Callisthene fasciculata Mart. (pau-jacaré), Dipteryx alata Vogel (barú), Guazuma ulmifolia Lam. (mutamba) and Magonia pubescens A. St.-Hil. (tingui). This study is the first to record a wide floristic list of this important region of central Brazil.