Mutation stimulation is a viable and well-established breeding approach for plants enhancement, induction bioactive compounds, and genetic variety generation to create novel plant mutants. The objective of this study was to mutate Gardenia jasminoides Ellis plant by gamma radiation (0, 15, 20, 25, 30, 35, and 40 Gy) as well as to determine the morphological and biochemical parameters of in vitro and in vivo treated plants. Consequently, the study has to detect the genetic diversity of the obtained mutants. The results indicated that gardenia plant irradiated with gamma at dose of 15 Gy showed the greatest growth rate. In addition, the maximum contents of chlorophyll (a, b) and carotenoids were detected in shootlets mutated with 15 Gy of gamma rays. Whereas, the highest total phenolic (TPC) and flavonoid contents (TFC), anthocyanin amounts, and antioxidant capacity were found in shootlets exposed to 40 Gy of gamma rays in vitro and in vivo. Furthermore, the suspected genetic variation in Gardenia jasminoides Ellis after treatment with γ-irradiation was evaluated using inter-simple sequence repeat (ISSR) analysis which produced a total of 98 bands, with the percentage of polymorphic bands ranging from 30 to 60%. According to the similarity matrix, the dendrogram was constructed into three clusters. Consequently, the results concluded that γ-irradiation of Gardenia jasminoides Ellis has given a sufficient number of induced mutations. Subsequently, the ISSR analysis has provided a powerful molecular marker for identifying mutants. Therefore, the in vitro  radiation-induced mutation could be a useful technique for assisting breeding programs for new Gardenia cultivars.