This study aims to isolate and identify taxonomic characterization of endophytic fungi from bitter melon in Guangdong province, China. A total of 1172 endophytic fungi are isolated from roots, stems, leaves, flowers, and fruits of healthy plants, and they are classified to 49 taxa based on morphological and molecular features. The results show that endophytic fungi from bitter melon plants exhibit high biodiversity. Arthrinium aureum, A. marii, A. sphaerospermum, Corynascus verrucosus, Curvularia borreriae and C. protuberate have not been recorded in any plants in China. Basidiomycetous endophytes, such as Ceratobasidium sp., and C. cornigerum, are reported in this study for the first time in bitter melon. According to the results of phylogenetic analysis, the rDNA ITS (Internal Transcribed Spacer) sequences can successfully separate species, such as F. solani, F. kyushuense, C. verrucosus, C. globosum, E. rostratum, C. brachyspora, C. verruculosus, C. affinis, P. bougainvilleicola, P. longicolla, P. glabrae, P. verruculosum, P. oxalicum, P. citrinum, P. chermesinum, P. glomerata, A. fumigatus and A. japonicas. Some isolates belonging to A. alternata, C. gloeosporioides, C. cladosporioides, C. brasiliense, C. convolutum, F. proliferatum, F. oxysporum, F. verticillioides, F. equiseti, F. camptoceras and Xylaria, however, require the analysis of others molecular markers to provide better taxonomic resolution. Molecular analyses of rDNA ITS sequences are useful for identification and classification of endophytes of bitter melon, but it is desirable to consider an integrated approach, such as molecular phylogeny, host, colony growth rate, symptoms, and shape of conidia.