Soybean [Glycine max (L.) Merr.] is the world’s most widely grown protein/oilseed crop and provides about 70% of global protein meal and 53% of vegetable oil in the United States. Soybean seed oil contains five major fatty acids, from which palmitic acid and stearic acid are two saturated fatty acids, oleic acid improves oxidative stability and linolenic acid is an essential fatty acid for human health. Soybean seed protein and oil are two important quality indices for soybean germplasm breeding. Soluble carbohydrates present in soybean meal provide metabolizable energy in livestock feed. To develop soybean germplasm with improved seed composition traits, it is important to discover novel source of seed fatty acid, protein, and carbohydrates traits. This dissertation aims to develop novel functional genomic technology coupled with an integrated approach for facilitating molecular soybean breeding. In this study, the first objective is to develop a high-throughput TILLING (Targeting Induced Local Lesions IN Genomes) by Target Capture Sequencing (TbyTCS) technology to improve the efficiency of discovering mutations in soybean. The robustness of this technology underlies the high yield of true mutations in genes controlling complex traits in soybean. Soybean mutagenized lines with modified fatty acids composition have been successfully developed to meet the different needs of end users. Altered fatty acids phenotypes have been associated with induced mutations in 3-ketoacyl-acyl carrier protein (ACP) synthase II (GmKASII), Delta-9-stearoyl-acyl carrier protein desaturase (GmSACPD), omega-6 fatty acid desaturase 2 (GmFAD2), and omega-3 fatty acid desaturase (GmFAD3) genes identified through TbyTCS. The second objective is to characterize the soybean acyl-ACP thioesterase gene family through a comprehensive analysis. The additional members have been discovered belonging to 16:0-ACP fatty acid thioesterase (GmFATB) gene family. The mutations at oleoyl-ACP fatty acid thioesterase (GmFATA1A) have been revealed to result in the high seed oleic acid content. The novel alleles of GmFATB genes have also been identified to confer low palmitic acid and high oleic acid phenotypes in soybean seeds. The third objective is to assess the phenotypic variations and correlation among seed composition traits in mutagenized soybean populations. Correlation analyses have been conducted among soybean carbohydrates, protein, and oil content of soybean mutagenized populations and germplasm lines. Chemical mutagenesis played an essential role in soybean breeding to generate novel and desired seed composition traits.