In Slovenia, the European chestnut (Castanea sativa Mill.) is threatened to collapse due to the attack of the chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu). In an experiment of vegetative propagation, between 2015 and 2016, we have included different interesting genotypes of chestnut from different regions of Slovenia that were damaged due to the attack. Our goal was to obtain healthy planting material. Explants for the design of in vitro culture were obtained from herded buds on the shoots, which we left 10 – 14 days in the water. The resulting shoots were surface-disinfected with 1.66 % dichloroisocyanuric acid. From disinfected shoots we prepared node explants which were then inoculated on an MS-½NO3 multiplication medium. We monitored the contamination of culture and the emergence of viable, vitrified and degraded shoots. We have found that between genotypes there exist statistically significant differences in the regeneration of viable, hyperhydric (vitrified) and degraded shoots. Genotypes which were statistically different from the remaining genotypes were genotypes Š2 from 2015 and Š4 from 2016. We confirmed that the year of sampling in the regeneration of vital shoots has impact on regeneration and that the interaction genotype:year is statistically significant, while the interaction genotype:subcultivation is not statistically significant. In vitro rooting was unsuccessful, because we were not able to obtain vital shoots with roots. We found that the greatest impact on micropropagation has genotype and its physiological condition.