Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most yield-limiting diseases of soybean worldwide. Releasing SCN-resistant cultivars are the primary basis for controlling SCN. Identification of parent lines showing major resistant loci and desirable agronomic characteristics are prerequisite for breeding resistant cultivars. In this study, a total of 487 soybean accessions were genotyped using two KASP markers that targeted to two resistance loci rhg1 and Rhg4. The accessions, which were identified harboring resistant alleles, were further subjected for resistance inoculated with race-2, race-4, race-5 and race-X12 in the greenhouse. Twenty accessions were characterized simultaneously harboring rhg1 and Rhg4 loci, while two accessions was found with the Rhg4 locus. Testing for SCN resistance suggested that one accession showed moderate resistant to three races and five accessions showed resistant or moderately resistant to two races. One accession was resistant and four accessions were moderately resistant to race-2. Two accessions showed moderately resistant to race-4. Four accessions showed resistant and fourteen moderately resistant to race-5. However, these accessions were turned out to be moderately susceptible or susceptible against race-X12. Collectively, by taking use of 487 soybean accessions this study identified 20 accessions that harbored two major resistant loci and showed desirable agronomic characteristics, might serve as elite parental lines in breeding for resistant cultivars via pyramiding rhg1 and Rhg4.