This study investigated the induction of genetic variability in rice through tissue culture techniques using hydrogen peroxide (H2O2). Two rice accessions underwent varying soak durations (12 and 24 hours) and concentrations (0%, 20%, 40%, 60%, 80%, and 100% H2O2). Following soaking, the seeds were inoculated into Murashige and Skoog (MS) media in a completely randomized design with three replications under in vitro conditions. Phenotypic traits, including the number of shoots, shoot length, number of roots, and root length, were analyzed through ANOVA with a significance threshold of ≤ 0.05. Results indicated significant differences among the treated plantlets and the control group, with specific treatments demonstrating superior performance in various physical parameters. Notably, the highest mean number of shoots (2.06) was observed in the control group (12 hours) for accession NGB00789, followed by 2.00 in the control group (12 hours) for NGB00792. Conversely, the lowest mean value (0.17) was recorded for NGB00792 soaked with 100% H2O2 for 12 hours. Regarding shoot length, NGB00789 soaked with 100% H2O2 for 12 hours exhibited the highest mean value (4.44 cm), while NGB00792 soaked with 60% H2O2 for 24 hours reached a mean value of 4.36 cm. For the number of roots, the highest mean value (6.67) was observed in NGB00789 under the control condition, followed by 6.22 in NGB00792, also in the control group. The lowest mean value (0.56) was recorded in NGB00792 soaked with 100% H2O2 for 12 hours. Concerning root length, the highest mean value (6.01 cm) was achieved in NGB00792 treated with 60% H2O2 for 24 hours, while NGB00792 soaked with 20% H2O2 for 12 hours reached a mean value of 5.99 cm. The lowest mean value (0.32 cm) was recorded in NGB00789 treated with 80% H2O2 for 24 hours. In conclusion, this study not only sheds light on the practical applications of hydrogen peroxide in rice tissue culture but also underscores the necessity for further research in this domain to fully harness the potential of mutagenesis as a tool for crop improvement.