Abstract: The dry soybean seeds (Glycine max, ‘Williams 82’) were irradiated by ¹²C⁶⁺ and γ rays. The contemporary biological effects were investigated comprehensively by measuring a series of indexes of growth and development, photosynthetic physiology and genetic polymorphism of M₁ generation seedlings, as well as the cluster analysis. The results showed that radiation treatment had inhibitory effects on length, width and area of the unifoliate leaf, length of the first internode at 14-day, plant height at 28-day, photosynthetic pigment content, chlorophyll fluorescence parameters (ETR(I), Y(I)), while it promoted the ratio of length to width of unifoliate leaf, length of the second internode length, chlorophyll fluorescence parameters Y(NA), and intercellular CO₂ concentration (Ci). Analysis of the genetic polymorphism of unifoliate leaf based on molecular marker technology revealed that Nei's genetic distance, Shannon index, and polymorphism rate increased in M₁ generation after radiation treatment. All the above indicators can be used as valid indicators to assess the response of soybean to both types of ionizing radiation. In addition, within a certain dose range, survival rate, the length of the first internode length, and plant height at 14-day can be used for γ-radiation effect assessment; while net photosynthetic rate (Pn), maximum photosynthetic rate (Pm), transpiration ratio (Tr), and stomatal conductance (Gs) can be used for carbon ion beam radiation effect assessment This study provides a reference for the selection of suitable parameters for soybean mutation breeding by radiation.