The phenotypic and genotypic studies on species responsible for grey mould disease, Botrytis cinerea and Botrytis pseudocinerea, are usually carried out on purified strains. Purification is classically achieved with the isolation of a single spore. This method entails fastidious and time-consuming steps and requires a good level of experience of the manipulator. An alternative method is based on the isolation of hyphal fragments under the stereomicroscope. Both the methods are widely used, but their efficiency has never been formally quantified. The two methods were compared for the purification of mixes of strains with known genotypic profiles. To assess the efficiency of the purification, the resulting isolates were characterized with micro-satellite markers and verified that each represented a single genotype. Both the methods had equally high efficiencies, up to 93–95%. The advantage of the simplified method is that it is much less time consuming than the single spore isolation (2 vs. 19 days) and requires less technical experience.