Strawberry anthracnose, caused by Colletotrichum species, is a major fungal disease threatening strawberry industry in Sichuan Province of southwestern China. However, research on identification of Colletotrichum species associated with strawberry anthracnose in Sichuan remains uncovered. In this study, 73 representative Colletotrichum strains were isolated from diseased leaves, stolons, petioles and crowns of 11 major strawberry planting localities in Sichuan Province. Based on morphological characteristics and multi-loci phylogenetic analysis, the Colletotrichum strains were identified as three distinct species: Colletotrichum fructicola (53 strains, 72.60%), C. siamense (17 strains, 23.29%) and C. gloeosporioides sensu stricto (3 stains, 4.11%). Among them, C. fructicola was the most ubiquitous and dominant species, whereas, C. gloeosporioides sensu stricto only restricted in Chongzhou. Importantly, our pathogenicity tests showed that C. fructicola and C. siamense can infect both leaves and stolons, while C. gloeosporioides sensu stricto was only pathogenic to leaves. Interestingly, although the sexual stage of C. siamense was not observed in this study, it still exhibited the strongest virulence to strawberry as compared to C. gloeosporioides sensu stricto and C. fructicola. Overall, this is the first study to characterize Colletotrichum species causing strawberry anthracnose and evaluate their pathogenicity in Sichuan Province of southwestern China, which will provide a better strategy on accurate diagnosis and management on anthracnose disease on strawberry.