13S globulin is one of the major storage proteins in buckwheat seeds. This study investigated the 13S globulin gene sequence by PCR specific amplification and sequencing using 6 species of genus Fagopyrum, a total of 44 collections including cultivated and wild species of F. esculentum and F. tataricum, F. pilus, F. zuogongense, F. gracilipes, F. urophyllum. Variance analysis of ultra-conserved segments of 13S globulin gene sequence indicated that a total of 285 arrangement sites (excluding deletion and insertion loci) were identified including 24 invariable (monomorphic) sites and 261 polymorphic (segregating) sites (198 parsimony informative sites and 63 singleton variable sites), and a total of 503 mutations on Eta site were isolated in the whole sequence. The intraspecific genetic diversity of 13S globulin gene sequence for the wild species of common buckwheat was obviously higher than that of the cultivated species of common buckwheat, and for the wild species of tartary buckwheat was slightly higher than that of the cultivated species of tartary buckwheat,cultivated species of tartary buckwheat was higher than common buckwheat. However, a lower intraspecific genetic diversity of 13S globulin gene sequence for was found in wild species of tartary buckwheat when compared to wild species of common buckwheat. These results implied that the distinct genetic diversity of 13S globulin gene sequence between F. esculentum and F. tataricum might be due to the fact that only a few wild type groups were domesticated during evolution as well as the different reproductive patterns. artificial domestication leaded to a severe narrow of genetic diversity of 13S globulin gene sequence in common buckwheat with cross-pollination characteristics during evolution. Cluster analysis indicated that the cultivated species of F. esculentum was closely clustered with its wild species, and further grouped with F. zuogongense, while the cultivated species of F. tataricum was closely clustered with its wild species, and further grouped with F. pilus. The highly ultra-conserved gene sequence of 13S globulin of F. gracilipes and F. urophyllum was identified, implying a close relationship between F. gracilipes and F. urophyllum. These results provide the theoretical basis for research of genetic diversity and evolutionary relationship of genus Fagopyrum.