Soybean is the primary oilseed crop in the United States, with significant industrial value. Understanding the molecular mechanisms of heat stress tolerance in soybean is critical for developing stress-resistant cultivars. Current knowledge about the role of fatty acid desaturases (FADs) in modulating membrane fluidity under abiotic stress prompted this investigation into the impact of mutations in the FAD genes on seed germination from heat-stressed plants. In soybean, exposure to heat stress during anthesis is known to significantly reduce seed germination. In silico expression analysis indicated high levels of expression of the soybean FAD2 and FAD3 genes in leaves. Therefore, a detailed expression analysis of these genes was conducted using qRT-PCR from leaf tissue. Generally, downregulation of these genes was observed in the mutants; however, two genes, FAD3A and FAD2-3, showed a more than 2-fold increase in expression in six out of ten mutants under heat stress. This upregulation was particularly pronounced (7-fold) in mutant S17CR-170. Correlation analysis revealed a positive correlation (0.5) between the expression level of FAD3A, FAD3B, FAD3C, and FAD2-3 and the decline in germination from heat-stressed plants. This suggests these FAD genes may act as negative regulators of germination under heat stress conditions.