<p><i>Calibrachoa hybrida</i> has high ornamental value due to its abundant flower color and long blooming period. This study investigates an efficient <i>in vitro</i> propagation system for <i>Calibrachoa</i> using leaf explants from twelve genotypes. The optimization of culture media for callus formation and plant regeneration of different <i>Calibrachoa</i> genotypes showed differences. The 100% callus induction rate was present using Murashige and Skoog medium supplemented with 0.5 mg·L^-1 6-benzylaminopurine (BAP) and 0.1 mg·L^-1 α-naphthaleneacetic acid (NAA). Significant genotype-dependent variations were observed in callus growth and differentiation characteristics, with genotypes ‘JDJ’ and ‘CBH’ exhibiting robust callus formation while ‘ZS’ and ‘CBQF’ displayed limited growth. The study also explored the effects of various plant growth regulators on shoot proliferation, revealing that increased BAP concentrations negatively impacted shoot development, whereas kinetin (KT) significantly enhanced proliferation rates. Shoot regeneration was achieved in four out of twelve genotypes. The three genotypes ‘XH’, ‘CBMH’, and ‘CBXGH’ were prone to producing adventitious shoot through direct and indirect organogenesis, with the highest regeneration rate of 75.8% in ‘CBMH’. However, the genotype ‘JDJ’ regenerated only via indirect organogenesis, and the regeneration rate was 10%, even on the optimal medium, which contained 2.0 mg·L^-1 KT and 0.1 mg·L^-1 NAA. A total of 2 mg·L^-1 KT and 0.1 mg·L^-1 NAA were conducive to rooting. These findings highlight the importance of genetic background in optimizing tissue culture techniques for <i>Calibrachoa</i>, providing valuable insights for future research and applications in ornamental plant propagation.</p>