Botrytis cinerea Pers. is a necrotrophic and polyphageous pathogen economically important on soft fruits and crops producing severe agricultural losses. The Chilean strawberry, Fragaria chiloensis (L.) P. Mill. is one of the wild parents of commercial strawberry and has the capacity to tolerate the infection from the pathogenic fungus. A study was performed to make a genetic analysis of wild and cultivated individuals collected from three different regions of Central South Chile and compare the Botrytis infection performance. The dominant ISSR markers were used to generate the genetic fingerprinting and the native samples were contrasted against the commercial Fragaria x ananassa Duch. The samples from Contulmo presented the lowest diversity within population and the highest diversity between populations, but at the same time showed the highest tolerance to Botrytis infection. Infected leaves and fruits from F. x ananassa showed 3.6% of the area covered by the fungus, three days after inoculation, while the plants from Contulmo showed 3.3% of the area covered by the pathogen, five days after inoculation. The plants from Contulmo were classified as highly tolerant (HT), tolerant (T), moderately tolerant (MT), and susceptible (S) where 50% of plants belonged to the group of tolerant. The results, highly significant (p