Plant-parasitic nematode species are one of the greatest threats to cause serious yield loss throughout soybean production regions of the United States. Soybean [Glycine max (L.) Merr.] cultivars of different maturity groups carry known resistance to soybean cyst nematode (Heterodera glycines, SCN) at rgh1 and Rhg4 loci. Continuous use of these genes caused SCN population shifts and overcoming host resistance over time. Lack of genetic diversity created a bottleneck in development of new cultivars containing novel SCN resistance genes. Additional sources of resistance to southern root-knot nematode (Meloido-gyne incognita, SRKN) and reniform nematode (Rotylenchlus reniformis, RN) could result in supplemental protection of soybean yield. This research focused on determining known and new sources of SCN resistance in 1220 soybean accessions divided into two groups: (i) early-maturity groups (000-II) screened with two SCN populations, and (ii) medium-maturity groups (III-V) screened with six SCN populations. Based on the initial screening results, a subset of 76 accessions was selected for further phenotyping with SCN, SRKN, and RN populations and supplemented by genotyping using molecular markers. The results revealed that 56 lines were resistant to two nematode species in various combinations, and 12 lines were resistant to all three nematode species. Moreover, potential new sources of resistance-carrying genes other than rhg1 and Rhg4 loci were pinpointed. Interestingly, RN resistance highly correlated with rhg1 locus suggesting a common gene or tight linkage. Identified resistant soybean accessions provide promising materials for discovery of novel genes to support cultivar development.