<p>Enhancing photosynthesis is an important approach to improve crop yields. Photosynthesis, as a key factor determining crop yield, is an important approach to increasing crop production and addressing global food security issues. Improving its efficiency is crucial in this regard. However, traditional photosynthetic phenotyping has long been a bottleneck in crop breeding due to time-consuming data collection. In this study, we simultaneously measured the spectral reflectance and the net photosynthetic rate (Pn) of soybean leaves to develop a high-precision model for estimating Pn based on hyperspectral data. By applying this model, we evaluated Pn in 219 soybean materials. A multi-environment genome-wide association study (GWAS) based on multi-environmental prediction Pn was carried out using the 3VmrMLM method, and 24 significant quantitative trait loci (QTLs) and four suggestive QTLs were identified. Among them, 24 QTLs overlapped with multiple previously reported QTL related to photosynthesis, chlorophyll content, quality, etc., or with genes related to key agronomic traits such as yield. Additionally, four new QTLs were discovered, and four candidate genes potentially associated with Pn were identified. Further, haplotype analysis identified their optimal haplotypes. This study presents a robust and nondestructive hyperspectral model for estimating the photosynthetic rate in soybeans, which is successfully applied to genetic analysis, yielding stable and biologically meaningful results. The approach offers an effective means to explore the genetic basis of photosynthesis and provides a solid theoretical foundation for large-scale, monitoring of soybean photosynthetic physiology.</p>