Abstract: An extensive survey of anthracnose in mango caused by Colletotrichum spp. was conducted in seven locations from western, central and eastern, Florida, USA and five locations from western and southern, Puerto Rico. Morphological, serological and molecular characteristics of 183 Colletotrichum spp. isolates was determined. Ninety three percent of the isolates from Puerto Rico and Florida, USA were identified as C. gloeosporioides, while only 5% (eight isolates) as C. acutatum. Another 2% (3 isolates) from the same host were negative for both species. Pathogenicity tests conducted on detached mango leaves showed that both species were pathogenic. Necrotic lesions produced orange to salmon conidial masses on acervuli 7 days after inoculation. Colonies of isolates producing aerial mycelia were white, gray and/or dark gray, often with conidial masses on acidified potato dextrose agar. Conidia of isolates were hyaline, one-celled, ovoid to oblong, straight or slightly curved and ranged from 12 to 20 x 3.5 to 6 μm for C. gloeosporioides and 8 to 13 x 2 to 5 μm for C. acutatum. ELISA and PCR assays were used complementary to morphological results and confirmed C. acutatum causing anthracnose in mango`s flower, peduncles and immature fruit in Homestead, Florida. Colletotrichum acutatum was neither found during this survey and has never been reported on mango in Puerto Rico. Colletotrichum spp. identification is essential in the development of control strategies of anthracnose disease because of differences within species to benomyl and azoxystrobin. Other fungi isolated from necrotic lesions in mango were identified as Phomopsis sp., Cladosporium sp., Curvularia sp., Fusarium pallidoroseum, Pestalotiopsis sp., Alternaria infectoria and Nigrospora sp. Pathogenicity tests also indicated that A. infectoria, F. pallidoroseum and Pestalotiopsis sp. were pathogenic to detached mango leaves under laboratory conditions.