Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 μM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.