The Sierra Mazateca is located in the northern mountainous region of Oaxaca, Mexico, between the Valley of Tehuacán-Cuicatlán and the Gulf Coastal Plains of Veracruz. It is part of the more extensive Sierra Madre de Oaxaca, a priority region for biological research and conservation efforts because of its high levels of biodiversity. A floristic study was conducted in the highlands of the Sierra Mazateca (at altitudes of ca. 1,000–2,750 m) between September 1999 and April 2002, with the objective of producing an inventory of the vascular plants found in this region. Cloud forests are the predominant vegetation type in the highland areas, but due to widespread changes in land use, these are found in different levels of succession. This contribution presents a general description of the sampled area and a checklist of the vascular flora collected during this study that includes 648 species distributed among 136 families and 389 genera. The five most species-rich angiosperm families found in the region are: Asteraceae, Orchidaceae, Rubiaceae, Melastomataceae, and Piperaceae, while the largest fern family is Polypodiaceae.