Anthracnose caused by Colletotrichum spp. is a serious disease of strawberry. The etiology of anthracnose of strawberry is complex, and several Colletotrichum spp. have been regarded as causal agents. In the present study, multilocus (actin, β-tubulin, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase) phylogenetic analysis revealed that 100 isolates of Colletotrichum associated with anthracnose of strawberry in central China belong to five species. In total, 97 isolates were identified belonging to the Colletotrichum gloeosporioides species complex, with C. murrayae, C. gloeosporioides, C. fructicola, and C. aenigma accounting for 81, 8, 4, and 4% of the total isolates, respectively. Three isolates belonging to the C. acutatum complex were identified as C. nymphaeae. On inoculated strawberry plants, isolates of C. fructicola and C. murrayae species showed strong pathogenicity to both leaves and petioles of strawberry, with plant mortality 30 days after inoculation of 77.8 and 55.6%, respectively. C. gloeosporioides, C. aenigma, and C. nymphaeae showed strong pathogenicity to leaves but weak pathogenicity to petioles, with plant mortality 30 days after inoculation of 5.6, 16.7, and 11.1%, respectively. The five species were divided into four classes based on their maximum growth temperatures. Isolates of C. murrayae and C. gloeosporioides were more tolerant to high temperature (>34°C) than isolates of other species, followed by C. fructicola and C. aenigma. Isolates of C. nymphaeae, which are only distributed in areas of higher altitude (1,100 m), were highly sensitive to higher temperature. These results indicate that pathogenicity and adaptation to temperature are important factors in the distribution of Colletotrichum spp. on strawberry plants. This research may increase our understanding of how Colletotrichum spp. emerge and spread to geographical regions with different latitudes or elevations.