Strawberry is economically important. Anthracnose caused by Colletotrichum spp. has been a serious threat to this crop globally. To detect and distinguish latent Colletotrichum spp. infection on asymptomatic plants at an affordable cost is crucial for this disease control and the sustainability of strawberry industry. Loop-mediated isothermal amplification (LAMP) assay is superior for the higher sensitiveness, efficiency, and lower cost than other methods for pathogen diagnosis. In this study, six previously reported barcodes were evaluated against DNA templates from strawberry fungal pathogens including six Colletotrichum species and five beyond this genus, and further verified in 40 Colletotrichum isolates. Based on the discernibility revealed, a LAMP assay was developed for the diagnosis of Colletotrichum spp. by designing six primers recognizing the conserved regions in β-tubulin 2 gene from 13 Colletotrichum species of C. gloeosporioides and C. acutatum complexes. The specificity and accuracy of LAMP assay was tested against six Colletotrichum species of two complexes, with a detection sensitivity of 100 pg/μL genomic DNA. Current LAMP assay beginning with a 10 min plant lysis enabled a direct and quick diagnosis of Colletotrichum spp. in strawberry within one hour. Followed by PCR using primers specific to ApMat, Marker 2, and Marker 1, this assay could specifically differentiate Colletotrichum species, at least for the dominant species C. fructicola and C. siamense in China, realizing a diagnosis of latent infection at a species level. Current LAMP-PCR combined protocol allows for a sensitive and efficient detection and differentiation of latent Colletotrichum infection on strawberry, which will be useful for disease management and monitoring pathogen population.