Crops harvesting ensure human survival on earth. However, sustainable crop production is challenging because of continuous biotic stresses caused by different pests (e.g., nematodes, bacteria, fungi, viruses, and insects). Soybean (Glycine max) is an important legume crop supplying more than half of the world’s proteins, oil, and fats. It is challenged by the Soybean Cyst Nematode (SCN), Heterodera glycines (HG), the most devastating pest worldwide. Currently, the widely used soybean cultivars are losing resistance due to rapid evolution of SCN populations. Developing novel and diverse soybean cultivars resistant to SCN is in urgent need. In this study, we used the soybean wild relative (Glycine soja; G. soja from now onwards), to study the genetic basis of plant chemical defense to SCN.We combined multiple approaches including metabolomics and genetics-, to investigate the molecular mechanism(s) underlying nematode resistance in wild soybean (G. soja) toward the long-term goal to develop new SCN resistant varieties. Specifically, for the first time, we integrated metabolomics, gene expression, and hairy root transformation analyses to identify the genetic basis conferring broad-spectrum resistance to two rarely studied SCN races (SCN5, HG type 2.5.7 and SCN2, HG type 1.2.5.7).The outcome of the present study showed involvement of phenyl-propanoid pathway genes and metabolites in SCN2 and SCN5 resistance. Comparison of the metabolic profiles among SCN resistant (S54) and susceptible (S67) wild soybean genotypes showed clear differences, mirroring the solid part of phenolic acid conjugates (1-O-4-hydroxybenzoyl-β-D-glucose ester, 1-O-3, 4-dihydroxybenzoyl-β-D-glucose ester, 1-O-3, 4, 5-trihydroxybenzoyl-β-D-glucose ester, 1-O- 4-hydroxy-3-methoxybenzoyl-β-D-glucose ester, N-benzoyl-L-glutamic acid and 4-hydroxybenzaldehyde) in defense. The results also indicated that many isoflavonoids (daidzein, daidzin, malonyl daidzin, genistein, formononetin, iso-formononetin) might play important roles in SCN2 and SCN5 resistance. One of the candidate genes, hydroxyisoflavanone/isoflavone-4’-methyl transferase (HI4’OMT/I4’OMT), is a small gene family and a genotype-specific allele (H3) was found in the resistant genotype S54. Overexpression of this H3 allele in SCN susceptible soybean cultivar (Willimas 82) using well-developed soybean hairy-root transformation significantly inhibited the development of both SCN races, as well as decreased the SCN numbers, which suggested that this S54 genotype-specific allele could increase levels of SCN resistance. The results of this study provided a foundation toward the goal to develop novel soybean cultivars with enhanced SCN resistance.