Anthracnose caused by Colletotrichum spp., is one of the most serious diseases affecting tamarillo (Solanum betaceum) production in the Ecuadorian highlands. The objective of this study was to characterise Colletotrichum isolates obtained from tamarillo to clarify its taxonomic and phylogenetic position. Based on phenotypic and morphologic characterisation, the isolates of this study were consistently grouped within the Colletotrichum acutatum complex. Multilocus molecular phylogenetic analysis based on combined sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences using Bayesian probabilities, indicated that 16 of 20 isolates from Imbabura, Cotopaxi, Tungurahua, Bolivar, Chimborazo, Azuay and Loja provinces, belonged to C. tamarilloi. This study represents the first reported case of anthracnose of S. betaceum caused by C. tamarilloi in the Ecuadorian highlands. Interestingly, pathogenicity tests and multilocus molecular analysis revealed a new infraspecific and more aggressive species formed by four isolates obtained from the Pichincha province. These findings probably provided interesting information related to mutant isolates and a possible description of a new pathogenic species affecting tamarillo fruits.