Prunus cerasus L. 'Oblačinska' sour cherry germplasm was established in vitro directly from in situ plants on different explant collection dates, enabling quick clonal multiplication and introduction to a rootstock breeding program. Rosette initiation of four investigated genotypes was possible from November to April on the medium containing Schenk and Hildebrandt (SH) macroelements, Murashige and Skoog (MS) microelements, and vitamins supplemented with (in mg L-1) 6-benzyladenine (BA), 0.5; indole-3-butyric acid (IBA), 0.01; gibberellic acid (GA 3), 0.1; citric acid, 10; and L-ascorbic acid, 10. The lowest percent of contamination was noted in November and December, when dormant buds were used as an explant source, and the highest percentage was found with actively growing shoot tips. The elongation phase of rosettes initiated from dormant buds remained a major obstacle. Survival rates of shoot tips obtained in April were high and subsequent growth more prominent. An increasing index of multiplication from 1.5 to 1.9 was noted on Driver and Kuniyuki walnut medium (DKW) with 0.8 mg L-1 BA and 0.01 mg L-1 IBA in the 'OV 32' genotype. Rooting percentages of 71.3% and 81.3% were achieved in 'OV 17' and 'OV 32' genotypes, respectively, on half-strength MS medium with 1 mg L-1 IBA.