Strawberry anthracnose caused by Colletotrichum spp. is considered one of the most serious and destructive disease of strawberry worldwide. Weeds, as possible hosts of the pathogen, could have a role as reservoir and inoculum source for strawberry infections. To prove this hypothesis, symptomless weeds were collected in strawberry fields showing anthracnose symptoms in Iran. Ten isolates with Colletotrichum-like colonies were recovered from symptomless Amaranthus viridis L., Sonchus oleraceus L., Convolvulus arvensis L., Lactuca serriola L. and Adonis aestivalis L. plants as endophyte. The isolates were identified as C. nymphaeae, based on a combination of morphological and sequence data of TUB and GADPH genes. This identification was further validated using Rep-PCR fingerprinting analysis, which produces species-specific DNA fingerprints and unveils inter and intra variation of the species examined in this study. Moreover, rep-PCR marker was used to reveal accurate taxonomic position of Colletorichum spp. causing strawberry anthracnose belonging to C. acutatum complex, including C. acutatum sensu stricto, C. fiorinae, C. godetiae, C. nymphaeae, C. salicis and C. simmondsii. The C. nymphaeae isolates originating from symptomless weeds confirmed their pathogenicity on detached strawberry leaves, proving that weeds in strawberry field may have a role as source of inoculum. However, further studies are necessary to quantify their real contributions to anthracnose epidemics in strawberry fields. In this study, above-mentioned weeds are reported as the possible hosts of C. nymphaeae for the first time all over the world.