Sweet chestnut is a devastated species in Croatia, since it has been plagued by chestnut blight and ink disease, and in recent years new pests. Planting new trees of more resistant genotypes would be an important undertake. New seedlings can be produced generatively, but because of allogamy, the planting material in this case is not uniform. Micropropagation as a vegetative propagation method implies a solution. The objective of this study was to establish the protocol for micropropagation of sweet chestnut (Castanea sativa Mill.). Shoot tips from axillary buds of young root shoots were isolated under stereomicroscope and placed on establishing medium. As explants either microshoots (0.5 – 1 cm) or nodal segments were used. The influence of cytokinins 6-benzylaminopurine and zeatin on microprogation efficacy was tested. The effects of different agars and different concentrations of gibberellic acid were also tested. The nodal segments had significantly higher number of shoots per explant than the microshoots. However, the length of the longest shoot and the average length of shoots was significantly higher on the microshoots. Explants developed significantly much better when propagated on MS medium 1.5 mg/L 6-benzylaminopurine (BAP) then on MS medium with 1.5 mg/L zeatin. The highest number of shoots per explant (2.7) was obtained from nodal segments on the MS medium with 1.5 mg/L BAP. The largest length of the longest shoot (9.5 mm) and the largest average shoots length (8.9 mm) were obtained on the microshoots on MS medium with 1.5 mg/L BAP.