Publisher's version/PDFDue to the high polymorphisms between synthetic hexaploid wheat (SHW) and common wheat, SHW has been widely used in genetic studies. The transferability of simple sequence repeats (SSR) among common wheat and its donor species, Triticum turgidum and Aegilops tauschii, and their SHW suggested the possibility that some SSRs, specific for a single locus in common wheat, might appear in two or more loci in SHWs. This is an important genetic issue when using synthetic hexaploid wheat population and SSR for mapping. However, it is largely ignored and never empirically well verified. The present study addressed this issue by using the well-studied SSR marker Xgwm261 as an example. The Xgwm261 produced a 192 bp fragment specific to chromosome 2D in common wheat Chinese Spring, but generated a 176 bp fragment in the D genome of Ae. tauschii AS60. Chromosomal location and DNA sequence data revealed that the 176 bp fragment also donated by 2B chromosome of durum wheat Langdon. These results indicated that although a single 176 bp fragment was appeared in synthetic hexaploid wheat Syn-SAU-5 between Langdon and AS60, the fragment contained two different loci, one from chromosome 2D of AS60 and the other from 2B of Langdon which were confirmed by the segregating analysis of SSR Xgwm261 in 185 plants from a F2 population between Syn-SAU-5 and Chinese Spring. If Xgwm261 in Syn-SAU-5 was considered as a single locus in genetic analysis, distorted segregation or incorrect conclusions would be yielded. A proposed strategy to avoid this problem is to include SHW’s parental T. turgidum and Ae. tauschii in SSR analysis as control for polymorphism detection