Sexual propagation is not suitable for production of true to type plants for commercial cultivation of selected cultivars due to the having heterozygous characteristics. Rubus species are highly heterozygous and therefore when they are propagated by seeds the high plant variability is obtained. Micropropagation methods became very important and have many advantages compared to the classical propagation methods. In Turkey, blackberries are commonly propagated by hard and soft wood cuttings, by layering and bush division. Propagation by layers requires rather large area for a layered, and weed control among the layers is a problem. However, propagation by cuttings is connected with important problem in inadequate rooting, especially in 'Chester Thornless' cultivar, which is one of the most commercially grown cultivar. In addition, limited number of plants can be propagated using classical propagation method and thus demand of growers for the plants can not be satisfied. Optimized and highly efficient regeneration protocols are also important for genetic transformation for improving of new cultivars. In this study we focused on optimization of protocol for in vitro propagation of 'Chester Thornless' blackberry cultivar using nodal segments. For this purpose, explants were cultured on Woody Plant Medium (WPM) supplemented with combination of plant growth regulators BAP (1.5, 2, 3 mg/l) with NAA (0, 0.1, 0.2, 0.4 mg/l) to detect the best multiplication medium. According to the obtained results, the highest proliferation rate was obtained for cultivar 'Chester Thornless' that produced 9.66 shoots on a medium containing 2 mg/I BAP+0.2 mg/I IBA combination. GA(3) effects on multiplication rates were also studied and generally the highest rates obtained by while GA3 were not used. For the rooting, different concentrations of IBA and NAA (0, 0.1, 0.2 and 0.4 mg/I) were compared. A concentration of 0.4 mg/l NAA gave the greatest number of roots and maximum root length.