Soybean is an important food crop that contains high amounts of isoflavones. However, due to the expression of multiple genes, different soybean seeds have different isoflavone compositions. The underlying mechanisms for this complexity remain unknown. In this study, we identified potential differentially expressed genes (DEGs) in two wild soybean cultivars, ZYD7068 (high isoflavone) and ZYD7194 (low isoflavone), at different seed developmental stages using RNA-seq technology and compared their differences in isoflavone content. A total of 1067 and 6479 differentially metabolized genes were identified at R6 and R8 stages, respectively. Subsequent analysis of the KEGG pathway revealed that three of these differential metabolized genes were involved in the Isoflavonoid biosynthesis and Flavone and flavonol biosynthesis at the R6 stage. A total of 80 TF genes encoding differential expression of MYB, bZIP, and WRKY were identified in A1 vs. B1 and A3 vs. B3. Eight differentially expressed genes were identified in duplicates at both stages, and three genes showed the same expression trend at both stages. To confirm the results of RNA-seq, qRT-PCR was performed to analyze the expression of the six identified differentially expressed genes (DEGs). The results of qRT-PCR were consistent with the results of RNA-seq. We found that four genes (Glyma.13G173300, Glyma.13G173600, Glyma.14G103100, and Glyma.17G158900) may be involved in the positive regulation of isoflavone synthesis, while two genes (Glyma.04G036700 and Glyma.19G030500) may be involved in the negative regulation of isoflavone synthesis. These findings suggest that the observed difference in isoflavone levels between the two cultivars may be attributable to the differential expression of these six genes at later stages of seed development.