Transcription factor binding sites (TFBSs) are the functional elements that determine the cooperation of several transcription factors required for specific spatial-temporal expression patterns. We conducted de novo motif discovery of the cis-regulatory elements among a set of co-regulated genes which were selected from six reported Affymetrix Soybean GeneChip microarray studies involving a compatible soybean [Glycine max (L.) Merr.]–soybean cyst nematode (SCN, Heterodera glycines Ichinohe) interaction. These selected genes have been shown to be induced significantly in susceptible soybean infected with SCN. Collectively, a total of 18 induced genes were selected as candidate genes for promoter analysis. For de novo motif search, we applied 7 computational tools as representatives of three distinct algorithm classes: enumeration (Weeder, MDScan), deterministic optimization (MEME, BioProspector) and probabilistic optimization (MotifSampler, W-AlignACE), as well as employing a synthetic approach (SCOPE). We applied a synthetic strategy to look for overlapping motif regions which were detected by at least 4 out of the 7 bioinformatics tools. A total of 111 overlapping motif regions were discovered. Thereafter, we conducted biological functional analysis of the detected motif regions including the surrounding nucleotides as well as motifs alone in transgenic soybean hairy roots generated via Agrobacterium rhizogenes-mediated genetic transformation. The inducibility of each motif region or motif itself was evaluated by co-localization of the gain-of-function of a red fluorescent protein (RFP) reporter gene and the presence of SCN in transgenic hairy roots. First, we selected 7 out of the 18 promoter regions for biological functional tests and found that 2 promoters are SCN-inducible at the early stage of SCN infection. Second, the inducibility of the 17 detected motif regions from these two promoters were tested and 64.7 % (11 out of 17) detected motif regions are SCN-inducible at the early stage of SCN infection. Last, the 11 early SCN-inducible motif regions were down-selected to further examine the elements for the core region of these early SCN-inducible motifs. As a result, at least 11 de novo core motifs were discovered experimentally. Taken together, these results demonstrate that this synthetic strategy to examine cis-regulatory elements for overlapping motif regions is a valid and effective approach for de novo motif discovery in the soybean genome.