The most important objectives of soybean (Glycine max (L.) Merr.) breeding are to increase oil content and to improve oil quality. Although the biochemical processes of oil accumulation in oilseeds are very clear, knowledge of their underlying genetic mechanisms and regulation is limited. We performed RNA-Seq of soybean seeds from six accessions with high, medium and low seed oil contents. Through comparative genome analysis, promoter-enrichment study, and protein–protein interaction (PPI) analysis, 80 lipid-metabolism-related genes and 31 transcription factors were detected. The pathways of fatty acid elongation, desaturation and export from plastid (P = 2.53E-4), and triacylglycerol biosynthesis (P = 2.31E-8), were significantly over-represented in accessions with high total oil content. Further, in an integration analysis of RNA-Seq and a genome-wide association study (GWAS) database, 62 candidate genes were found to be associated with seed oil content, 73 with oleic acid content, and 83 with linolenic acid content. Of these, 60 genes were found to be involved mainly in metabolism of lipids (25), carbohydrates (24), and amino acids (11). Thirty are known oil-synthesis-related genes; LOX1, CYP93D1 and GPT2 for oil content, SAD and FAD2 for oleic acid, and FAD2, CYP89A6 and GPT2 for linolenic acid were detected twice. There were 22 genes found to be associated with at least two oil-related traits, and of 154 pairs of PPIs, two genes for each pair of 95 PPIs (62%) were found to be associated with various oil-related traits, indicating the genetic foundations of oil-related traits. Three transcription factor genes were found to be associated with oil-related traits: HRE2 (Glyma.10G016500), ERF12 (Glyma.13G236600) and WRKY6 (Glyma.15G110300). This study provides an efficient strategy for further discovery of mechanisms of oil composition and accumulation.