This work aimed to evaluate the genetic diversity of Botrytis cinerea in Chile and to determine whether the two genetically different groups transposa and vacuma, described in France, are present in the country. Isolates collected in Chile from grapes, tomato, kiwifruit and blueberry were analysed using molecular markers. We developed a PCR test to identify the two groups of B. cinerea, transposa (with the transposable elements Boty and Flipper) and vacuma (with neither). As described in France, both kind of isolates were found to be present and sympatric in Chile. Isolates containing the transposable element Boty alone (boty isolates) were also detected. The frequencies of transposa, boty and vacuma isolates were significantly different on kiwifruit compared to grapes, tomato and blueberry. RAPD analysis revealed a high degree of genetic diversity, with no widespread clonal lineages. Dendrograms, analysis of molecular variance and Fst values revealed the existence of genetic differentiation in our sample between isolates from the different host plants. PCR-RFLP markers also showed that isolates sampled from grapes and tomato were genetically differentiated. Additional sampling is required to confirm these findings.