Purple seed stain (PSS) of soybean (Glycine max (L.) Merr.) is a prevalent seed disease. It results in poor seed quality and reduced seed lot market grade, and thus undermines value of soybean worldwide. The objectives of this research were to evaluate the reaction of selected soybean genotypes collected from 15 countries representing maturity groups (MGs) III, IV, and V to PSS, and to identify new sources of resistance to PSS based on three years of evaluation of natural field infection by Cercospora spp. in the Mississippi Delta of the U. S. In this study, 42 soybean genotypes were evaluated in 2010, 2011, and 2012. Seventeen lines including six MG III (PI 88490, PI 504488, PI 417361, PI 548298, PI 437482, and PI 578486), seven MG IV (PI 404173, PI 346308, PI 355070, PI 416779, PI 80479, PI 346307, and PI 264555), and four MG V (PI 417567, PI 417420, PI 381659, and PI 407749) genotypes had significantly lower percent seed infection by Cercospora spp. than the susceptible checks and other genotypes evaluated (P ≤ 0.05). These genotypes of soybean can be used in developing soybean cultivars or germplasm lines with resistance to PSS and for genetic mapping of PSS resistance genes. In addition, among these 17 lines with different levels of resistance to PSS, nine soybean genotypes (PI 417361, PI 504488, PI 88490, PI 346308, PI 416779, PI 417567, PI 381659, PI 417567, and PI 407749) were previously reported as resistant to Phomopsis seed decay. Therefore, they could be useful in breeding programs to develop soybean cultivars with improved resistance to both seed diseases.