Anthracnose disease is a major limiting factor to papaya production worldwide. Accurate identification of the pathogens responsible for this disease is important to developing disease management strategies. One hundred and three (103) isolates of Colletotrichum were collected from infected papaya fruits cvs. Red lady and Tainung No. 2 - F1 hybrid in Trinidad. Of all isolates, 79% were C. gloeosporioides and 21% were C. truncatum. Spore morphology, cultural characteristics, differential reaction to benomyl in addition to ITS1 and β-tubulin gene sequence comparisons unequivocally identified and separated the two species of Colletotrichum. Certain characteristics enabled discrimination between the two species and may be used for provisional identification of these species isolated from papaya. Isolates of C. gloeosporioides grew at a significantly faster rate than those of C. truncatum. C. gloeosporioides isolates were sensitive to benomyl at 1.0 μg/ml, but C. truncatum isolates were resistant. Pathogenicity tests revealed that Colletotrichum species and papaya cultivar had no significant effect on lesion diameter. In cross-infection studies, isolates of C. gloeosporioides from mango and C. truncatum from sweet pepper were able to infect and cause symptoms in wounded mature papaya fruit under controlled conditions. There was no evidence of anthracnose infection in seeds of infected fruits based on the results of growing-on tests. Phylogenetic analyses were based on comparisons of ITS1 and β-tubulin gene sequences. Both neighbor-joining and maximum parsimony methods resolved all Colletotrichum isolates from papaya into species-specific clusters with high bootstrap support. Additionally, a pair of species-specific primers were developed (Ct-TUB-F/R) which allowed reliable detection of C. truncatum isolates from papaya.