Between 2009 and 2012, 118 soybean [Glycine max (L.) Merr.] accessions from the USDA Soybean Germplasm Collection were screened for resistance to soybean rust (Phakopsora pachyrhizi) at up to five locations in the southeastern United States. In 2009, plant introductions (PIs) from maturity groups III through IX were evaluated for relative disease severity and intensity of sporulation from uredinia compared with 12 susceptible cultivars from the same range of maturity groups. Resistance evaluations were based primarily on disease severity and intensity of sporulation from rust pustules. To assess resistance at several nurseries, a rust index score was calculated from the severity and sporulation ratings. Many of the PIs were moderately to highly resistant at the 2009 locations between Alabama and South Carolina, but the P. pachyrhizi population in Bossier City, LA, was virulent on most of those accessions. The 2011 rating data from Quincy, FL, indicated an increase in the virulence of the pathogen there since 2009, and this trend was observed again in 2012. In contrast, many of the same PIs developed substantially less soybean rust in Attapulgus, GA, in 2012. Despite the comparatively greater disease that many accessions had in Louisiana in 2009 and in Quincy in 2011 and 2012, at least 78 PIs were resistant in Georgia in 2012, and 20 of those were at least moderately resistant in both Florida and Georgia that year. No accessions were immune to rust at all of the nurseries, but PI 200492 (Rpp1), PI 547875 (a backcross line with Rpp1), and PI 567102B (Rpp6) were the most resistant of the accessions with named resistance genes and were among the most resistant accessions overall. Among the most resistant accessions with unknown resistance genes, PI 416826A, PI 417125, PI 567034, and PI 567104B consistently had effective levels of resistance in different locations and years. Information about the most resistant PIs and their reactions to soybean rust infection across years and locations will be useful for the development of rust‐resistant soybean cultivars in the United States.