Genetic disturbance in wild populations of Medaka (Oryzias latipes) has resulted from the introduction of populations originating from commercial strains. To clarify the genetic composition of orange-red type and wild type commercial strains of O. latipes (‘himedaka’ and ‘kuromedaka’, respectively), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analyses of the mitochondrial cytochrome b gene were conducted for Medaka populations cultured in 2 fish farms and others purchased from 21 aquarium fish shops. All ‘himedaka’ populations showed a high frequency of the B27 mitotype, an original mitotype of ‘himedaka’, and a low frequency of the B1a mitotype, an introduced mitotype from wild populations in Okayama or Nara Prefectures. ‘Kuromedaka’ populations, originating from cultured populations, were characterized primarily by the B27 and B1a mitotypes, being a result of hybridization between wild populations and ‘himedaka’. All ‘kuromedaka’ populations originating from wild populations had their own native mitotypes, although some also had the B1a mitotype, which was inferred as being of ‘himedaka’ origin.