The development of resistant soybean varieties is an efficient way to manage soybean rust in Brazil. To date, resistant varieties effective in Brazil have been limited due to the presence of highly virulent rust populations. This study aimed to identify effective resistant varieties/resistance genes in selected soybean genotypes against a highly virulent Brazilian rust population, BRP-2. Sixty-six soybean genotypes, including 57 soybean rust-resistant/tolerant genotypes previously identified, were exposed to BRP-2 and their resistance was evaluated based on lesion characteristics. PI 587905 produced typical resistant and susceptible lesions and was excluded from the resistance classification. Hence, 47 (72.3%) of the 65 genotypes tested were classified as susceptible, 14 (21.5%) were classified as slightly resistant and 4 (6.2%) were classified as clearly resistant. Although 18 genotypes were placed into resistant categories, PI594767A was the only genotype on which neither uredinia nor urediniospores were produced in two weeks after inoculation. In addition, urediniospores were observed in all 16 varieties carrying one of the five known resistance genes (Rpp1-Rpp5) and in one genotype in which two resistance genes, Rpp2 and Rpp4, were pyramided. Conversely, our screening of soybean genotypes by the rust population, BRP-2 inoculation revealed that two Chinese varieties, ‘Lu Pi Dou’ and ‘Hei Dou’, possessed a leaf-yellowing prevention characteristic. Leaf-yellowing, which might cause rapid defoliation leading to yield loss, was observed not only in susceptible varieties but also in a resistant variety ‘Shiranui’. The use of the resistance and/or tolerance identified in this study will assist in the development of resistant cultivars capable of enduring soybean rust in Brazil.