n this study, results of photomixotropic and photoautotrophic chestnut micropropagation are compared. The aim is to increase the quality of micropropagated plants as well as to reduce costs due to plant loss during acclimatization. A successful protocol has been developed for the proliferation phase without exogenous sucrose added to the medium that has been tested with 4 genotypes. For this, shoots were exposed to decreasing sugar levels for several months to ensure their viability in sucrose-free medium. In vitro rooting in photoautotrophic conditions was applied to 15 chestnut genotypes. Every genotype formed vigorous plants which were easy to acclimatize. More than 6000 shoots were rooted with a rooting and acclimatization average value of 71% for the best treatment. This result was better than the one obtained in conventional micropropagation.