Soybean (Glycine max (L.) Merr.) ranks as the second-largest crop by total production in the United States, despite its production experiencing significant constraints from plant pathogens, including those causing seedling diseases. Pythium irregulare Buisman stands out as a predominant driver of yield loss associated with the seedling disease complex. There is currently a lack of public or commercial varieties available to growers with adequate genetic resistance to manage this pathogen. To address the pressing need for germplasm resources and molecular markers associated with P. irregulare resistance, we conducted a screening of 208 genetically diverse soybean accessions from the United States Department of Agriculture Soybean Germplasm Collection (USDA-SGC) against two geographically and temporally distinct isolates under controlled greenhouse conditions. Disease severity was assessed through comparisons of the root weight and stand count ratios of inoculated plants to mock-inoculated controls. Employing linear mixed modeling, we identified ten accessions (PI 548520, PI 548360, PI 548362, PI 490766, PI 547459, PI 591511, PI 547460, PI 84946-2, PI 578503, FC 29333) with resistance significantly above the population average to one or both of two isolates originating from Ohio or Indiana. Previously curated genotyping data, publicly accessible via the SoyBase database, was subsequently utilized for conducting a genome-wide association study. This analysis led to the discovery of two significant marker–trait associations (MTAs) located on chromosomes 10 and 15 and accounting for 9.3% and 17.2% of the phenotypic variance, respectively. The resistant germplasm and MTAs uncovered through this study provide additional resources and tools for the genetic improvement of soybean resistance to seedling disease caused by P. irregulare.