The development of stress-resistant crops without compromising their yield and nutritional quality is one of the major challenges in plant biology. Plant breeders have made considerable effort in developing cultivars with high yield and tolerance to biotic and abiotic stresses. This research aimed to evaluate pre-breeding resources for favorable genes conferring good yield and resistance to biotic and abiotic stresses for rice varietal improvement. Two hundred forty diverse rice cultivars from top countries with which the United States imports and exports rice were selected from the Germplasm Resource Information Network (GRIN) collection, and these were characterized using molecular markers for various desirable traits. Among the cultivars, 16 were used as checks. The genes of various traits evaluated were grain number (Gn1a), panicle weight (WFP), cold stress tolerance (qSCT-11 and qCTS-12), anaerobic germination (AG1 and AG2), rice blast disease resistance (PiB, Pi-ta, Ptr, and Piz), wide compatibility (S5-n), and restoring ability (Rf4). Out of these 240 accessions, 192 have the Gn1a gene, 168 possess the qSCT-11 cold tolerance gene, 139 carry the Rf4 gene, 152 have the qCTS12 cold tolerance gene, 87 have blast resistance genes, and 44 possess the WC gene. None of the 240 accessions possess the WFP (Wealthy Farmer’s Panicle) gene. The selections with favorable genes can be used by breeders as potential donor parents for varietal improvement programs to produce new and promising cultivars with desired specific traits that may contribute towards alleviating global issues related to food security.