Mango (Mangifera indica), a fruit with high nutritional value is widely regarded as a most palatable fruit crop which is affected by a number of biological constraints mainly diseases. Anthracnose caused by Colletotrichum gloeosporioides belongs to order Melanconiales is the important post harvest disease which restricts the marketing of mango. In the present study, thirteen isolates obtained from various places of Tamil Nadu were collected and isolated by tissue segment approach and purified. The isolates were confirmed phenotypically using morphological characters. Molecular investigation like PCR assay using universal primers ITS1 and ITS4 produced amplicon size of 560bp. The isolates were also identified using genus specific and species specific primers which resulted in the amplicon size of 280bp and 380bp. As a result, The use of morphological and molecular approaches to characterise Mango anthracnose will be useful in identifying and managing the disease.