Chemical compounds in soybean (Glycine max (L.) Merr.) seed have great effects on the flavour and taste of soymilk. The 1-octen-3-ol content in soybean seed could produce sensory off-flavours (mushrooms, lavender, rose and hay flavour) during the processing of soymilk. The most effective way to reduce off-flavours of soymilk is the screening and utilisation of soybean cultivars with reduced 1-octen-3-ol content. However, genomic study of 1-octen-3-ol in soybean has not previously been reported. In the present work, a natural population of 90 diverse soybean accessions was used to dissect the genetic basis of 1-octen-3-ol content in soybean seed through genome-wide association analysis by using 26 822 single nucleotide polymorphisms (SNPs). Twenty-one novel quantitative trait nucleotides (QTNs) were thus identified to be associated with 1-octen-3-ol content in soybean seed. Among them, 13 QTNs overlapped with, or were located in, the linked regions of known QTNs for protein, oil and/or fatty acid content in soybean seed. The functional genes located in the 200-kb genomic region of each peak SNP were considered possible candidates related to 1-octen-3-ol, such as genes involved in amino acid metabolism, protein content, and hormone metabolism. The identified loci with beneficial alleles and the candidate genes may be valuable for improving the off-flavours of soymilk.