Water deficit stress continues to be one of the major factors limiting soybean yield [Glycine max (L.) Merr.], the world’s leading economic oilseed crop. One of the major benefits of legumes in agriculture is their capacity to symbiotically fix atmospheric nitrogen, thus reducing the need to use nitrogen fertilizers. In drought conditions, soybean not only suffers from reduced leaf area development and photosynthesis, but its symbiotic N2 fixation is especially vulnerable. Earlier experiments have shown that increases in shoot ureide concentrations are associated with inhibition of N2 fixation in nodules under water deficit stress conditions. A collection of 373 diverse soybean genotypes were grown in two years at two different sites (Columbia, Missouri and Stuttgart, Arkansas) and characterized for shoot ureide concentration. Our preliminary analysis revealed abundant phenotypic and genetic diversity in the studied population. The population structure results indicated that the 373 diverse soybean genotypes could be clustered into eight major subpopulations. Genome-wide association studies (GWAS) based on unified mixed model using 12K SNPs (minor allele frequency, MAF ≥ 5%) marker showed location specific SNPs for both sites. A few SNPs showed association for shoot ureide concentration for each location in both years and some were found associated in at least three environments. Some of the loci identified by GWAS were located in or close to previously reported quantitative trait loci mapped by linkage analysis for shoot ureide concentration in biparental populations. Our preliminary results identified new potential quantitative trait loci (QTLs) for ureide dynamics and identified soybean genotypes for further investigation of N2 fixation-based drought tolerance.