The filamentous ascomycete Botrytis cinerea is one of the most studied models for understanding the necrotrophic behaviour of phytopathogenic fungi. The genomes of two strains of B. cinerea have been sequenced (B05.10 and T4), which may contribute to elucidating the virulence polymorphism in this fungus. In this study, both strains were genetically modified in order to construct recipient strains designed to target genes that are hard to knock out. Deletions of BcKu70 gene in B05.10 strain and BcKu80 gene in T4 strain both affected the nonhomologous end-joining (NHEJ) DNA repair mechanism. NHEJ is responsible for the ectopic integration of gene replacement cassettes during fungal transformation and leads to a lower frequency of homologous recombination (HR). Ku deficiencies in B. cinerea did not disturb in vitro or in planta growth, but clearly improved HR efficiency for the putative sesquiterpene cyclase-encoding gene Cnd15, which was hard to knock out in a wild-type strain.