China has introduced 3218 soybean accessions from 26 countries such as the United States, Russia and Japan, and some of them have been carried out soybean cyst nematode (SCN), soybean mosaic virus (SMV) and salinity tolerance resistance evaluation. However, the systematic genotyping of these accessions has not been reported yet. In this study, five robust functional markers have been developed for KASP assays, three SCN loci (rhg1, Rhg4, SCN3-11) and salinity tolerance gene (GmSALT3) included. A total of 1489 introduced soybean accessions were genotyped by these markers with high-throughput assay as well as a SCAR marker (SCN11) which is related to soybean mosaic virus resistance. The results showed that there were 1084 accessions detected with favorable alleles; where accessions detected with resistant alleles at three loci were as much as 19, including three pyramiding SCN genes (rhg1, Rhg4, SCN3-11) which were Peking type and seven pyramiding SCN and SMV , two pyramiding SCN and salinity favorable alleles, as well as seven pyramiding SCN, SMV and salinity favorable alleles; and accessions detected with four favorable alleles were as much as nine accessions, including six pyramiding SCN and SMV resistance alleles, one accession detected with SCN and salinity tolerance and two detected with SCN, SMV and salinity favorable alleles, eight detected with all the favorable alleles in this study. Among the elite accessions mentioned above, it has been proved that 44 accessions resistant to SCN, SMV-3 or tolerant to salinity. Among the 36 accessions with three or more favorable alleles, 52.78% had been reported of one or two characteristics. Among the accessions without resistance or tolerance alleles, it has been reported that 93 accessions were tolerant to salinity or resistant to SMV-3, where new resistance or tolerance genes could be found. Screening out the accessions with high-throughput SNP detection assays for resistance and tolerance alleles in soybean provides information for their further phenotyping, screening and breeding.