Large numbers of single nucleotide polymorphism (SNP) markers are now available for a number of crop species. However, the high-throughput methods for multiplexing SNP assays are untested in complex genomes, such as soybean, that have a high proportion of paralogous genes. The Illumina GoldenGate assay is capable of multiplexing from 96 to 1,536 SNPs in a single reaction over a 3-day period. We tested the GoldenGate assay in soybean to determine the success rate of converting verified SNPs into working assays. A custom 384-SNP GoldenGate assay was designed using SNPs that had been discovered through the resequencing of five diverse accessions that are the parents of three recombinant inbred line (RIL) mapping populations. The 384 SNPs that were selected for this custom assay were predicted to segregate in one or more of the RIL mapping populations. Allelic data were successfully generated for 89% of the SNP loci (342 of the 384) when it was used in the three RIL mapping populations, indicating that the complex nature of the soybean genome had little impact on conversion of the discovered SNPs into usable assays. In addition, 80% of the 342 mapped SNPs had a minor allele frequency >10% when this assay was used on a diverse sample of Asian landrace germplasm accessions. The high success rate of the GoldenGate assay makes this a useful technique for quickly creating high density genetic maps in species where SNP markers are rapidly becoming available.