Brown blight disease caused by Colletotrichum species is a common and serious foliar disease of tea (Camellia sinensis). Fungal isolates from several tea plantations causing typical brown blight symptoms were identified as belonging to the Colletotrichum acutatum species complex and the Colletotrichum gloeosporioides species complex based on morphological characteristics as well as DNA analysis of the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Colletotrichum acutatum, a new causal agent associated with C. sinensis, showed high phenotypic and genotypic diversity compared with the more commonly reported C. gloeosporioides. Phylogenetic analysis derived from individual and combined ITS and GAPDH sequences clearly clustered C. acutatum and C. gloeosporioides into separate species. Pathogenicity tests validated that both species were causal agents of tea brown blight disease and were highly pathogenic to tea leaves. However, the two groups of C. gloeosporioides with low levels of variability within their ITS and GAPDH regions differed in their virulence. This study reports for the first time the characterization of C. acutatum and C. gloeosporioides causing brown blight disease on tea (Camellia sinensis (L.) O. Kuntze) in China.