Individual plants of a Japanese onion variety Sapporo-ki, characterized by the occasional occurrence of male sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male fertile and the Jones’ cytoplasmic male sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox I, cox II, cob, atpA and atp9 genes as probes revealed the presence of two classes of mtDNA variation within a population of Sapporo-ki: out of 41 plants examined, 19 contained mtDNA typical of male fertile plants and 22 individuals contained mtDNA typical of the Jones’ CMS genotype. Our results thus indicate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.