Biological nitrogen fixation (BNF) is a fundamental process in soybean, positively related to high productivity and protein content and negatively related to environmental stress, both biotic and abiotic. To obtain high yields, soybean producers tend to apply chemical N fertilizers that increase the production cost, while causing ecological damage. Our goal in the present study is to develop soybean cultivars with high BNF capacity to support green agricultural systems and enhance sustainability. In accordance with our first-year proposal, we created a panel of 200 soybean accessions from 20 different countries that were genetically diverse for BNF according to information collected from an exhaustive literature and database review. Data for traits related to BNF were collected under greenhouse conditions using a protocol previously developed in our lab and combined with SNP marker data. Statistical analysis helped us to select two lines with high BNF, yield potential, and protein content that will be used for trait introgression. Additionally, a genome-wide association study revealed SNPs associated with BNF controlling traits that could be used as biomarkers for facilitating the selection of soybean varieties with high N fixation efficiency. Overall, our results allow us to continue our efforts toward the development of new germplasm material with significantly lower N demand, improved crop performance, and seeds rich in protein.