Silymarin is a constitutive secondary metabolite extracted fromSilybum marianumseeds with wide therapeutic and medical effects. Gamma irradiation could be a powerful tool for obtaining promising mutated genotypes of S. marianum with high contents of flavonolignans. The transcript levels of the chalcone synthase genes (CHS1, CHS2, and CHS3), encoded for silymarin, in response to two different gamma irradiation doses (200 and 600GY) were determined using qRT-PCR. Also, Molecular isolation and characterization of chalcone synthase genes and its encoded protein were investigated via bioinformatics tools. Results of qRT-PCR confirmed that exposure of S. marianum seeds to gamma irradiation up-regulated the expression of the CHS1, CHS2 and CHS3 genes which could be positively correlated with increasing of silymarin content in the fruits. In conclusion, the increasing of flavonolignans content conflicts a great contribution in the field of medicine.