The selection of genetically stable and physiologically uniform apple rootstocks is a critical prerequisite for the successful establishment of intensive orchards under arid and semi-arid environmental conditions. This study evaluated the genetic diversity, propagation efficiency, and clonal fidelity of widely used apple rootstocks (M.9, MM.106, and MM.111) through the integration of microclonal propagation and molecular genetic validation under the soil–climatic conditions of the Samarkand region. Axillary bud explants were cultured on optimized in vitro media, and regeneration performance was assessed during multiplication, rooting, and acclimatization stages, revealing significant genotype-dependent differences. The highest shoot multiplication coefficient was recorded in M.9 (4.6 ± 0.3 shoots per explant), followed by MM.106 (3.8 ± 0.2) and MM.111 (3.1 ± 0.2), while rooting success ranged from 74.2% to 82.4% and acclimatization survival rates exceeded 76–83% across genotypes, confirming the effectiveness of the applied micropropagation protocol under regional conditions. Genetic diversity among rootstocks was assessed using SSR markers, which revealed clear polymorphism and distinct genetic clustering corresponding to vigor classes and breeding backgrounds, with the mean number of alleles per locus ranging from 6.2 in M.9 to 9.1 in MM.111 and polymorphic information content values between 0.68 and 0.81. To evaluate genetic fidelity, ISSR and SCoT marker systems were applied to compare donor plants and in vitro-derived clones, and the analyses revealed predominantly monomorphic banding patterns with genetic similarity coefficients ranging from 0.98 to 1.00, indicating minimal or no detectable somaclonal variation. The strong agreement between morphophysiological performance indicators and multilocus molecular marker data confirms that axillary bud-based micropropagation preserves genome stability and produces true-to-type clones suitable for further propagation and micrografting. Overall, the integration of regionally optimized in vitro propagation protocols with comprehensive molecular validation provides a reliable framework for producing uniform, pathogen-free, and micrografting-ready apple rootstocks adapted to challenging environmental conditions, supporting sustainable orchard establishment and long-term productivity of apple cultivation systems in Central Asia.