Brassica napus L. is one of the most important oilseed crops in China and plays a critical role in ensuring the national supply of edible vegetable oils. However, compared with high photosynthetic efficiency crops such as maize and rice, rapeseed exhibits relatively low light energy utilization efficiency, typically less than 1%, whereas rice and maize can reach approximately 1.5% and more than 2%, respectively. Improving photosynthetic efficiency is therefore considered an essential pathway toward achieving high yield and environmentally sustainable rapeseed breeding. To systematically evaluate the photosynthetic performance of diverse B. napus germplasm and identify elite accessions with high photosynthetic efficiency, a total of 245 accessions were assessed at the seedling stage. Nineteen photosynthesis-related traits, including gas exchange and chlorophyll fluorescence parameters, were measured. The results revealed significant genotypic variation in gas exchange characteristics, PSII energy conversion efficiency, and thermal dissipation capacity, indicating rich genetic diversity and promising potential for selection. Correlation analysis showed that stomatal conductance (Gs) and transpiration rate (Tr) in seedling leaves were positively correlated with the performance index based on absorbed light energy (PI_ABS), the complementary area above the O-J phase of the chlorophyll fluorescence rise curve (Ss), and shoot fresh weight (SFW), while negatively correlated with the specific energy flux for electron transport per reaction center (ETo/RC). These findings suggest that coordinated regulation of photosynthesis and water use efficiency plays a key role in biomass accumulation. Moreover, the significant positive correlations of SFW with PI_ABS and Ss indicate that chlorophyll fluorescence parameters can serve as effective indicators of photosynthetic efficiency and biomass accumulation potential in rapeseed seedlings. Principal component analysis (PCA) extracted six major components. The first principal component was predominantly associated with chlorophyll fluorescence indicators such as Fv/Fm, Pi_ABS, and Phi_Eo, highlighting the close relationship between PSII quantum efficiency and overall energy conversion capacity. The second component was mainly related to thermal dissipation factors including Ss and DIo/RC, while the third to sixth components reflected traits related to stomatal regulation, electron transport, chlorophyll content, and biomass accumulation, respectively. Based on the PCA results, a comprehensive evaluation index (D-value) was constructed. Cluster analysis of the D-values grouped the accessions into five categories: 35 high-efficiency types, 52 moderately high-efficiency types, 49 intermediate types, 88 moderately low-efficiency types, and 21 low-efficiency types. From these, 12 elite accessions with superior photosynthetic efficiency were identified. Furthermore, a stepwise regression model was developed to determine the relationship between the comprehensive D-value and key physiological indicators. The optimal regression equation was: D = 1.049X? + 0.854X? + 0.387X? + 0.691X? + 0.607X? + 0.164X? – 0.225 (R2 = 0.984), where the six independent variables (X? to X?) represent Mo, ABS/RC, Tr, Pi_ABS, Fv/Fm, and Ci, respectively. In conclusion, the comprehensive evaluation system developed in this study provides an effective framework for the preliminary assessment of photosynthetic efficiency in rapeseed at the seedling stage. The findings offer theoretical guidance and data support for the identification of high-efficiency germplasm and the breeding of high photosynthetic efficiency B. napus cultivars.