<p><i>Taxodium</i> hybrid ‘zhongshanshan’ is characterized by its tolerance to water and salt, resistance to wind, and disease resistance. As a result, it is extensively utilized in ecological protection and landscaping, which satisfies a large market demand. However, traditional breeding methods for <i>Taxodium</i> hybrid ‘zhongshanshan’ often require lengthy hybridization and screening processes. Therefore, to facilitate genetic improvement and accelerate the cultivation of new varieties, there is an urgent need to establish an efficient and stable genetic transformation system. In this study, we explored suitable culture medium and hormone conditions for their rooting, and first achieved rooting in DCR medium containing 0.4&#xa0;mg·L⁻¹ IBA and 0.3&#xa0;mg·L⁻¹ NAA. Callus was used as material to investigate the impact of antibiotics, including Geneticin (G418), Hygromycin (Hyg), Kanamycin (Kan), Cefotaxime Sodium (Cef), and Ampicillin (Amp) on their proliferation and differentiation. Results indicated that Kan 50–75 mg·L^− 1 served as the critical tolerance concentration for callus proliferation and differentiation. For G418, the suitable concentration was determined to be 70 mg·L^− 1 –90 mg·L^− 1, and the critical tolerance concentration for Hyg was 15–20 mg·L^− 1, respectively. Amp 300–400 mg·L^− 1 was the critical tolerance concentration for <i>Taxodium</i> hybrid ‘zhongshanshan’, and Cef did not inhibit the callus growth. Subsequently, co-culturing callus with <i>Agrobacterium</i> revealed that 70 mg·L^− 1 G418 combined with 400 mg·L^− 1 Cef had a better antibacterial effect, which can be considered the suitable selection concentrations for genetic transformation. The investigation of organogenesis optimization and antibiotic screening will lay a foundation for stable genetic transformation system.</p>