The fatty acid content plays a crucial role in the quality traits of soybeans. However, its content is easily affected by environmental and genetic factors. Therefore, conducting QTL mapping research on the fatty acid content in soybeans is of great significance for mining genes related to the regulation of soybean fatty acid content. In this study, a high-density genetic linkage map containing 761 SSR and InDel marker loci was constructed. The map length was 1475.7 cM, and the average distance between markers was 1.94 cM. In this study, the multiple QTL model (MQM) mapping method was used, and a total of 49 QTLs related to fatty acid content that existed in two or more environments were detected. By comparing with the QTL mapping results reported by previous studies, this study found that 9 QTLs overlapped with the previous research results. A total of 4 QTLs that could be stably inherited in 4 environments were detected. Analysis of these four QTL intervals finally screened out 5 candidate genes related to soybean fatty acid content. The results of this study provide new ideas for marker-assisted selection breeding and point out the direction for improving the quality traits of soybeans.