Tartary buckwheat is one of minor grain crops in China, which has rich nutrition and is an important source of natural rutin. It is an important aspect of present research to break through of breeding and create new germplasm in tartary buckwheat. The mutation library was constructed in “Heifengyihao” by EMS. The half lethal treatment of EMS mutagenesis was 1.2%, the effect of plentiful phenotype was recorded in M1 generation. 102 plants with obvious phenotypic variation, including leaf color, leaf shape, plant type and grain sizes, were obtained from M3 materials and the mutation rate was 3.85%. The rutin content of 1000 plants in mutants’ library was determined by HPLC, 2 mutant lines with high (rutin content >16 mg/g) and 5 lines of low rutin (rutin content <10 mg/g) were obtained respectively. Comparison of rutin synthesis-related genes through qRT-PCR showed a relative weaker relationship between FtCHS, FtF3H, Ft4CL, FtUFGT transcripts and rutin content mutation lines. However, it also found that FtFLS showed the higher expression level in line with higher rutin content, which reached 4.55 times of control. The screening of mutants enriched the resources of tartary buckwheat, innovated the new germplasm of tartary buckwheat, and provided material guarantee and technical support for the molecular basis research of tartary buckwheat metabolism.