Karyotype analysis and classification of buckwheat chromosomes were performed without chemical banding or staining using atomic force microscopy (AFM). Fagopyrum esculentum (common buckwheat) and Fagopyrum tartaricum (Tartarian buckwheat) chromosomes were isolated from root tissues using an enzymatic maceration technique and spread over a glass substrate. Air-dried chromosomes had a surface with ridges, and the height of common and tartary buckwheat were approximately 350 and 150 nm. Volumes of metaphase sets of buckwheat chromosomes were calculated using three-dimensional AFM measurements. Chromosomes were morphologically characterized by the size, volume, arm lengths, and ratios. The calculated volumes of the F. esculentum and F. tartaricum chromosomes were in the ranges of 1.08–2.09 μm3 and 0.49–0.78 μm3, respectively. The parameters such as the relative arm length, centromere position, and the chromosome volumes measured using AFM provide accurate karyomorphological classification by avoiding the subjective inconsistencies in banding patterns of conventional methods. The karyotype evolutionary trend indicates that F. esculentum is an ancient species compared to F. tartaricum. This is the first report of a cytological karyotype of buckwheat using AFM.