A major focus in cultivar development is increasing yield while maintaining or decreasing current fertilizer and irrigation inputs. The USDA-ARS National Small Grains Collection (NSGC) could be a valuable source of such traits, which may or may not be found in modern wheat varieties. In this study, 540 spring wheat accessions from the NSGC core subset, representing germplasm of diverse geographic origin, and 5 adapted check lines were evaluated for tolerance to nitrogen deficiency and water stress across three treatments: irrigated/normal nitrogen, terminal drought/normal nitrogen and terminal drought/low nitrogen. Agronomic and physiological traits and novel high-throughput canopy spectral reflectance indices were used to assess lines. To enable genome-wide association analysis, accessions were genotyped with the Illumina 9000 SNP platform, which identified 5029 polymorphic markers. High yielding accessions tolerant to nitrogen deficiency and water stress were identified. Q + K mixed model analyses using JMP Genomics statistical software revealed several significant marker-trait associations. Once consensus marker positions are available for the SNP markers, QTL mapping will be possible.