Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily-primed PCR (ap-PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap-PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap-PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β-tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap-PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.