The genetic diversity of four new species related to southwestern Sichuan buckwheats was examined using karyotypes, allozymes and intersimple sequence repeats (ISSR). Karyotype analysis showed that Fagopyrum wenchuanense, F. qiangcai and F. pugense are diploid (2n = 2x = 16) while F. crispatofolium is tetraploid (2n = 4x = 32). The karyotype evolutionary trend of buckwheat indicated that F. crispatofolium, F. gracilipes var. odontopterum and F. gracilipes seemed to be more advanced than F. wenchuanense, F. qiangcai, F. pugense, F. esculentum, F. cymosum and F. tataricumare. Three polymorphic enzyme systems were used for the examination of variation, including peroxidase, esterase and superoxide dismutase. Consistent banding patterns were obtained for esterase and superoxide dismutase, while peroxidase produced a large number of sharp bands. A total of 18 ISSR primers were selected for the analysis and showed high variations among the species. Allozymes and ISSR markers were utilized to estimate the genetic distance among accessions and to draw phylogenetic trees. Our data provide evidence of a high degree of genetic diversity among southwestern Sichuan buckwheats. In addition, both cultivars and wild types showed a high degree of divergence suggesting a complex domestication process in this crop. This study provides a better understanding of evolutionary mechanisms and genetic relationships in four new species of buckwheat.