The objective of this research was to identify SNPs and to develop an Illumina iSelect beadchip with over 50,000 soybean SNPs. A total of 498,921,777 reads 35-45bp in length were obtained from DNA sequence analysis of reduced representation libraries created from two wild and six cultivated soybeans. These reads were mapped to the soybean whole genome sequence and identified 209,903 SNPs. After applying several filters, a total of 146,161 SNPs were determined to be ideal candidates for Infinium II beadchip design. In order to equalize the distance between selected SNPs along each chromosome, increase assay success rate, and minimize SNPs with low minor allele frequency, an iteration selection algorithm based on a selection index was developed and was used to select 60,800 SNPs for Infinium beadchip design. Of the 60,800 SNPs, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes. In addition, 99 SNPs were targeted to unanchored sequence scaffolds. Of the 60,800 SNPs, a total of 52,041 passed Illumina’s manufacturing phase to produce the SoySNP50K iSelect SNP beadchip. Validation of the SoySNP50K chip with 96 landrace genotypes, 96 elite cultivars and 101 wild soybean accessions showed that 47,446 SNPs were polymorphic and could generate successful SNP allele calls. In addition, 40,747 of the 47,446 SNPs (85.9%) had minor allele frequencies ≥10% among the landraces, elite cultivars and the wild soybean accessions. A total of 749 and 17 candidate regions which may be associated with domestication and recent selection were identified, respectively.