Peach cultivars (’Elberta’, ‘Red Top’, and ’Dixie Red’) were studied for their in vitro adoptability and performance in producing virus-free plantlets. The thermotherapy method with increasing temperatures (25 °C to 37 °C) was applied for the elimination of famous peach infecting plum pox viruses (PPVs) and prunus necrotic ringspot virus (PNRSV), and the DS-ELISA test and RT-PCR technique were performed to confirm the production of virus-free microshoots. The application of 30 mg L⁻¹ of Fe-EDDHA treatment resulted in the best performance for culture establishment of all cultivars, where the best subsequent morphological performance in terms of branch and leaf numbers was recorded for the ’Dixie Red’ cultivar in MS medium, supplemented with 0.5 mgL⁻¹ of gibberellic acid (GA₃) and 0.5 mg L⁻¹ of 6-Benzylaminopurine (BAP). At the regeneration stage, the highest (26.96 mm) and lowest (18.43 mm) shoot lengths were obtained from the ’Dixie Red’ cultivar treated with GA₃ (2 mg L⁻¹) + thidiazuron (TDZ) (2.5 mg L⁻¹) and the ’Red Top’ cultivar treated with GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹), respectively. The leaf numbers were affected by the application of growth regulators, where the ’Elberta’ cultivar under GA₃ (2 mg L⁻¹) + TDZ (2.5 mg L⁻¹) treatment showed the highest numbers and the ’Red Top’ cultivar under GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹) showed the lowest mean values. The thermotherapy treatment and micropropagation of shoot tips resulted in 100% virus-free plantlets, as confirmed by both applied diagnostic methods. The result of the application of the rooting stage with growth regulators on ’Elberta’ plantlets showed the best performance (90%) in ½ MS medium supplemented with 0.5–1 mg L⁻¹ of IBA, which was significantly higher than the same treatment in MS medium. The obtained results should constitute the basis for further optimization of the multiplication and rooting of virus-free peach plantlets to be served for nurseries and planation orchards.