In vitro propagation of pistachio (Pistacia vera L.) faces multiple challenges due to strong genotype dependency, explant browning, and variable responses to plant growth regulators (PGRs). This study aimed to optimize different stages of micropropagation, including explant establishment, shoot proliferation, and root induction, using seedling explants of the cultivars "Tirmahi Ardakan," "Tirmahi Meybod," and "Haj-Abdollahi." After seed germination, explants were disinfected with mercuric chloride for 50, 100, and 200 seconds and cultured on MS medium. Shoot proliferation was tested using various concentrations of BA (0–4 mg L⁻¹) and IBA (0 and 0.01 mg L⁻¹), while rooting in "Tirmahi Ardakan" was evaluated using eight culture media supplemented with IBA and NAA. Results indicated that disinfection duration significantly affected explant browning, with the highest browning observed at 100 and 200 seconds (scores of 1.77 and 1.88 out of 5, respectively). During shoot proliferation, the highest number of shoots per explant (6.66) was obtained in "Tirmahi Ardakan" with 4 mg L⁻¹ BA + 0.01 mg L⁻¹ IBA, whereas the greatest shoot length (3.92 cm) occurred in "Haj-Abdollahi" with 0.1 mg L⁻¹ BA. In rooting, MS medium supplemented with 1.6 mg L⁻¹ thiamine + 1 mg L⁻¹ IBA and WPM with 1.5 mg L⁻¹ IBA produced the highest number of roots (4.5 roots per explant) and the longest roots (2.68 cm), respectively. These findings demonstrate that genotype-specific optimization of culture media and PGRs is critical for successful micropropagation of pistachio.