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Background: Anthracnose disease caused by the genus Colletotrichum is one of the crucial problems occurring in the field, along with postharvest diseases and affects mango quality in Thailand. In particular, the Nam Dork Mai See Tong cultivar, which is highly susceptible to the disease, is an important product for exportation. Methods: In this research, thirty-seven Colletotrichum species isolate were obtained from anthracnose disease in mango cv. Nam Dork Mai See Tong in three provinces in Thailand. Morphological studies and molecular techniques using species-specific primers were investigated; moreover, the diversity of pathogens was analyzed using PCR amplification of inter simple sequence repeats (ISSRs) with 6 primers, including pathogenicity tests. Result: Morphological studies and molecular detection with species-specific primers revealed that 32 isolates belonged to the C. gloeosporioides species complex and 5 isolates to the C. acutatum species complex. The genetic diversity of pathogens was analyzed. PCR amplification using 6 ISSR primers produced 35 polymorphic bands. These bands were used to construct UPGMA, in which cluster analysis divided the 37 isolates into 3 main groups and 8 subgroups at 61-73% Jaccard similarity coefficient with cophenetic correlation (r) = 0.6781. The ISSR technique showed the greatest genetic variation among isolates collected from different locations. Hence, a study based on ISSR markers was profitable to investigate the phylogenetic relationship of the genus Colletotrichum. Pathogenicity tests revealed that PC006 (Ca) and CS005 (Cg) showed the highest aggressiveness, with disease incidences of 84.74 and 80.90%, respectively. This study indicates that the diversity of pathogenic Colletotrichum species related to mango plantations in Thailand is increasing. [ABSTRACT FROM AUTHOR]