This study had the objective of determining the subculture number and the shoot basal diameter that produces the best rooting rate ex vitro of chestnut, Castanea sativa Mill., microshoots obtained via in vitro culturing. The plant material corresponded to microcuttings obtained from embryo cultures with between 7 and 12 subcultures in the proliferative stage, for which Driver and Kuniyuki Walnut (DKW) medium was used with the macronutrients reduced to a half and supplemented with 1 mg L-1 6-benzylaminopurine (BAP) and 0.1 mg L-1 indole-3-butiric acid (IBA). The quick induction rooting method was used, submerging the base of the microcuttings in a solution of 0.5 mg mL-1 of IBA for 15 min. For the rooting stage, pine bark:perlite (4:1, v/v) substrate was used, carrying out the evaluation of results at 20 days after culturing. The evaluated variables were survival rate (%), rooting rate (%), root number, root length (mm), callus presence and visual aspect of the rooting system. The results showed a decrease of rooting capacity as the subculture number increases. The factor microcutting diameter did not present significant differences with respect to the evaluated variables.