This study aimed to induce genetic variations through callus initiation and regeneration using leaves of two apple rootstocks (MM106 and Balady) cultured on MS medium supplemented with two chemical mutagens (EMS and NaN₃). Variations were genetically discriminated using biochemical (protein and isozymes) and molecular (RAPD-PCR) analyses. The percentage of callus survival was decreased due to the applied of both mutagens at 0.03 mg/l [54.0 and 55.5 %] in comparison with control [80.0 %], as well as, reduced the regenerated plants percentage. Vegetative parameters of the regenerated plantlets were affected by mutagens e.g., percentage of multiple shoots, number of leaves/plant and number of roots/plant. Effect of EMS at 0.01mg/l was increased some parameters if compared with control [5.50, 4.66, 6.00, 4.66, 4.50 and 3.16 %], respectively, for multiplication percentage, number of leaves and number of roots per plant. However, EMS and NaN₃ at 0.03 mg/l decreased all the vegetative parameters. Protein and isozymes (peroxidase and poly phenyl oxidase) analyses were used to studying genetic variation of gene expression.  Protein banding patterns of Balady and MM106 revealed a total number of five bands, one common band (monomorphic band) was detected (20.0% monomorphism). In addition, the remaining four bands were polymorphic (80.0% polymorphism). Few bands appeared or absent in control and some treatments are considered as specific molecular markers for these treatments. On the other hand, isozymes banding patterns represent differences in density of bands with different mutagenic concentrations compared with control. However, RAPD analysis had successfully generated reproducible polymorphic products. The generated profiles revealed levels of polymorphism in Balady rootstock. These appeared thirty eight total amplified fragments distributed between twenty were polymorphic (52.6%) and eighteen were monomorphic fragments. However, the total number of amplified fragments in MM106 rootstock and its treatments were thirty six which containing eighteen polymorphic fragments (50%).