Soybean Kunitz trypsin inhibitor (SKTI) extensively exists in cultivated and wild soybeans with many kinds of variants, which is often used as a genetic marker to study the origin and dissemination of soybeans. In this study, 10131 Chinese soybean landrace accessions were identified for SKTI types and a set of representative sample of 187 soybean landraces was analyzed to assess the genetic diversity and relationships of different SKTI type accessions using 20 nuclear SSR markers and 5 chloroplast SSR markers. Results showed that at the nuclear DNA level, the genetic diversity of the Tib type accessions was lower than that of the Tia type, but the genetic diversity at the chloroplast DNA level was just opposite; The cluster analysis showed that the Tib accessions from different regions tended to cluster together preferentially, indicating a close relationship among those Tib accessions. The Tib type soybean landraces in Gansu Province were rich in diversity of chloroplast haplotypes, and were principally comprised of type Ⅱ and Ⅲ. The soybean landraces with chloroplast haplotype Ⅱ in Gansu might be the main source of the soybeans with the same type in other provinces. Based on that Tib type soybean landraces in Gansu Province had a higher genetic diversity and a close relationship with those from other provinces, Gansu Province might be a center of the origin for Tib type soybeans in China.