Abstract: In this study, two related rice tartary buckwheat lines (F.tataricum, Guimiku 18-1 and Guiheimi 12), two perennial tartary buckwheat lines (F.tatari-cymosum, Guiduoku 003C and Guiduoku 60), two common buckwheat lines (F.esculentum, Guihongtian 2 and 1412-1), two thick-shell conventional tartary buckwheat lines (F.tataricum, Dingku 1 and Liuku 2017) were cultivated in Guiyang in autumn of 2018. The A.flavus was isolated and identified from shell and rice grain at maturation stage, and high performance liquid chromatography (HPLC) was used to detect the aflatoxin of AFB₁, AFB₂, AFG₁, AFG₂ in all A. flavus strains isolates from shells and grains. The results showed that there were no A.flavus colonies from the shell of all varieties and the rice grains of common buckwheat, convention tartary buckwheat, and perennial tartary buckwheat. A. flavus colonies were isolated only from rice grains of rice tartary buckwheat, and there were a total of 4 strains of A. flavus isolated. There were the incidence of A. flavus of 1.56% and 0.78% in mature seeds of Guimiku 18-1 and Guiheimi 12 respectively. The isolated strains morphology and sequencing results of ITS sequence amplification products were identical with those of A. flavus. The results of toxin test showed that there were significant differences in toxin contents among different varieties and in toxin production ability among different strains. Only rice tartary buckwheat were detected four toxins in all varieties, the maximum production of Guimiku 18-1 was AFB₁, (5.861±0.055) μg/kg, AFB₂ was the minimum (1.605±0.052) μg/kg, the maximum production of Guiheimi 12 was AFB₁ (14.475±0.533) μg/kg, AFB₂ was the minimum (3.393±0.151) μg/kg;The toxin content of rice grains was much higher than that of isolated strains;there were significant differences in toxin-producing capacity among isolated strains, The maximum production of AFT was (11.102±0.095) μg/kg and the minimum production of AFT was (1.794±0.024) μg/kg. These results suggested that the A. flavus source in rice tartary buckwheat rice grains may be from the direct infection of some rice grain exposure resulting from shell partly dehiscence. The above results could lay a foundation for the study of resistance breeding of A. flavus in buckwheat and the preservation, transportation and storage of buckwheat seeds.