Soybean seed isoflavones are a type of secondary metabolites that can provide health and nutrition benefits for humans.  In our previous study, a stable quantitative trait locus (QTL) qIF05-1 controlling the seed isoflavone content in soybean was detected on chromosome (Chr.) 05 in a recombinant inbred line (RIL) population from a cross of Huachun 2×Wayao.  In this study, the parental lines were re-sequenced using the Illumina Solexa System with deep coverage.  A total of 63,099 polymorphic long insertions and deletions (InDels) (≥15 bp) were identified between the parents Huachun 2 and Wayao.  The InDels were unevenly distributed on 20 chromosomes of soybean, varying from 1,826 in Chr. 12 to 4,544 in Chr. 18.  A total of 10,002 long InDels (15.85% of total) were located in genic regions, including 1,139 large-effect long InDels which resulted in truncated or elongated protein sequences.  In the qIF05-1 region, 68 long InDels were detected between the two parents.  Using a progeny recombination experiment and genotype analysis, the qIF05-1 locus was mapped into a 102.2 kb genomic region, and this region contained 12 genes.  By RNA-seq data analysis, genome sequence comparison and functional validation through ectopic expression in Arabidopsis thaliana, Glyma.05G208300 (described as GmEGL3), which is a basic helix-loop-helix (bHLH) transcription factor in plants, emerged as the most likely confirmed gene in qIF05-1.  These long InDels can be used as a type of complementary genetic method for QTL fine mapping, and they can facilitate genetic studies and molecular-assisted selection breeding in soybean.