Castanea sativa Mill. is a specie of agroforestry interest widely grown in Europe, its current production it is mainly focused on the extraction of a quality brown fruit, highly demanded in the gourmet industry. In Chile, this specie might be successfully grown due to the climate and lack of diseases that may affect it. However, there is not a propagation system that can provide a plant massification for the establishment of fruit orchards. Given this situation, the micropropagation is a possible alternative to apply since this method is extensively studied in this species. The propagation in vitro of Castanea sativa has created successful results in terms of introduction and multiplication. Nevertheless, the propagation in vitro of direct-producer varieties of brown fruit is still poorly studied and for this situation, in order to stablish a production system of a variety of plants of great interest is necessary to evaluate the effects of some variables that affects the in vitro culture of woody as it is the genotype and cytokine source. For this, six varieties of chestnut from Italy and France and two types of cytokines were studied to evaluate their effects on the parameters in vitro establishment as survival, contamination, formation of basal callus, axillary bud break, etc. The results indicate that in the same culture conditions the varieties with better response is Marrone Citta di Castello (CC) and Marrone Chiusa di Pesio (CP), in relation to the formation of basal callus and morphological reaction capacity, where the percentage of explants was greater than 80% and 85%, respectively, independent of the type of cytokinin used. Moreover, the amount of explants with axillary bud break was higher in the treatment with 0.5 mg L-1 of 6-benzylaminopurine (BAP) with 100% in CC and 55% in CP, being there significant differences with the obtained response in the treatment with 0.5 mg L-1 of Thidiazuron (TDZ), where 10% and 15% of explants with axillary bud break were observed, respectively. These results indicate that the genotype and cytokine source have influence on the response of in vitro establishment of nodal segments of chestnut variety, therefore, to establish a propagation system it is necessary to adjust a specific protocol for each variety.