This study evaluates the extent of micro grafting success of the Kalamata cultivar (Olea europaea L.) onto two rootstocks, Kronaki and Coratina, as well as examining the influence of various factors on micrografting outcomes. Additionally, the research investigates the impact of mineral composition in the nutrient medium on the in vitro development of these rootstocks. The findings demonstrate that, cultivars and growth medium significantly impact germination performance, the Kronaki cultivar exhibiting a higher germination rate (74%) compared to Coratina (62.5%). Moreover, the Murashige and Skoog (MS) medium outperformed the Olive Medium (OM) in terms of survival rates and shoot elongation. Specifically, MS medium facilitated greater shoot growth (4.85 cm on average) compared to OM medium (4.2 cm). Kronaki showed higher shoot lengths (5.20 cm) than Coratina cultivar (4.5 cm) on MS that superiority than OM medium. During acclimatization, Kronaki also exhibited higher survival rates (72.5%) compared to Coratina (59%). Genetic fidelity analysis using Inter-Simple Sequence Repeat (ISSR) markers indicated a high level of genetic stability, with 96% monomorphic bands and only 4% polymorphism observed. These results highlight the critical role of genotype and growth medium in optimizing micrografting success and maintaining genetic integrity in olive rootstock propagation.