The paper presents a protocol for micropropagation of Prunus sp. rootstocks included in the sweet and sour cherry breeding program. Germplasm diversity for rootstock breeding derives from natural populations, where conditions and biological vectors for systematic infection with viral diseases are constantly present. The establishment of aseptic culture depends primarily on the explant type, as all selections were collected from natural habitat. For nearly all investigated selections, dormant buds were the favored source, due to enabling rosette initiation in more than 58% cases. In P. cerasus L. selections, 100% contamination was noted when shoot tips were used as an explant source. Significant influence of the double-phase medium on the number and height of multiplied shoots was observed in the standard cherry rootstock, ‘Gisela 6’. For P. fruticosa Pall., selection ‘SV1’ and ‘SV2’, and P. cerasus ‘D6’ selection, the double-phase medium also had a significant effect on the height of multiplied shoots, when compared to solid DKW (Driver and Kuniyuki Walnut) medium. Genetic variability of selections within the investigated species resulted in variable plant rooting success. Adding Fe-EDDHA (Ethylenediamine di-2-hydroxy-phenyl acetate ferric) in the 200 mg l-1 concentration to the rooting medium significantly enhanced the percentage of rooted plants. The highest rooting percentage was noted for ‘Gisela 6’ and ‘D6’ genotype at 1 mgl-1 IBA (indole-3-butyric acid), while 0.8 mgl-1 was the optimum concentration for P. mahaleb L. ‘M1’ selection. P. fruticosa genotypes required significantly higher IBA concentration for rooting (2.5 and 3.5 mg l-1).