Winter hardiness is one of the main traits affecting survival, productivity and cultivar choice for important horticultural plants in a temperate climatic zone. Given the complexity of the trait and the often fluctuating winter weather, extensive field testing over many years would be required to gain reliable results. Testing for cold tolerance under controlled conditions to grade genotypes has been used in several plant breeding programs to identify superior genotypes of different species. The aim of the present study was to investigate cold tolerance of microshoots of different genotypes of the genera Prunus and Fragaria after 7, 14, 28 and 56 days of cold acclimation, by measuring ion leakage and evaluating the critical temperature (CT50) after freezing in vitro. Genotypes of Rosacea family demonstrated distinct patterns of CT50 change during cold acclimation. Cold acclimation for 56 days decreased the CT50 value by 1.3-2.0°C for Prunus microshots and by 0.8-2.1°C for Fragaria microshoots in comparison to nonacclimated plants. The results of cold acclimation and freezing treatments of Rosacea family plants in vitro show that for maximal cold hardiness, acclimation for 56 days or longer is required. Sour cherry (Prunus cerasus) cv. 'Orkolija', garden strawberry (Fragaria × ananassa) cv. 'Melody', Virginia strawberry (F. virginiana) and musk strawberry (F. moschata) were the most winterhardy after 56 days' cold acclimation. The data obtained in the study can be used to improve breeding and cryopreservation technologies of Rosacea plants, and lay the foundation for identification of genes responsible for efficient cold acclimation and low temperature tolerance.