SSR markers were used to analyze the genetic diversity and construct a DNA molecular ID database of 60 buckwheat germplasms in order to accurately identify the buckwheat germplasm resources in Guizhou province. The results showed that 16 pairs of stable and polymorphic primers were screened from 100 pairs of SSR primers. A total of 174 polymorphic bands were amplified from 60 germplasm studied. The mean value of Shannon’s information index， Nei’s diversity index， and polymorphic information index was 0.337， 0.206， and 0.693， respectively. It is indicated that the polymorphism of primers is good， which can effectively identify the genetic diversity of 60 buckwheat germplasms. When the Dice genetic similarity coefficient was 0.374， all materials tested were clustered into three groups including A， B， and C. When the Dice genetic similarity coefficient was 0.484， the Tartary buckwheat group （Group A） was further divided into two subgroups including A1 and A2. The results of clustering SSR amplification products validated by capillary electrophoresis and 8 polyacrylamide gel （PAGE） electrophoresis were consistent. It is indicated that the efficient SSR molecular markers developed can effectively identify the genetic diversity and can be used to construct DNA molecular ID cards of important buckwheat germplasm in Guizhou province.