Gentianaceae is a large plant family and is distributed worldwide. As the largest tribe in Gentianaceae, Gentianeae contains 939–968 species, and the Qinghai-Tibet Plateau and adjacent areas are the main centers of diversity for Gentianeae. Species from the Gentianeae are widely used in traditional Tibetan medicine. In this review, a systematic and constructive overview of the information on botany, ethnomedicinal usage, phytochemistry, and pharmacological properties of Gentianeae in Tibetan medicine is provided. The results of this study are based on a literature search, including electronic databases, books, websites, papers, and conference proceedings. Botanical studies showed that Gentianeae includes the subtribe Gentianeae and Swertiinae, and several new genera and taxa have been identified. Approximately 83 species from Gentianeae were used in Tibetan medicine, among which Gentiana and Swertia constituted the largest number of species with 42 and 24 species, respectively. The species from Gentianeae are mainly used as Bangjian (སྤང་རྒྱན།), Jieji (ཀྱི་ལྕེ།), Dida (ཏིག་ཏ།), and Ganggaqiong (གང་གྰཆུང་།) in Tibetan medicine with different clinical applications. More than 240 formulas were found containing Gentianeae species with different attending functions. Phytochemical studies showed that the main active components of Gentianeae species are iridoids, xanthones, flavonoids, and triterpenoids. The bioactivities of plants from Gentianeae include hepatic protection, upper respiratory tract protection, joint and bone protection, glucose regulation, antibacterial, antioxidant, anticancer, and antiviral effects. This review will provide a reference for future research on natural resource protection, plant-based drug development, and further clinical investigation.