Structural heterogeneity depicted as heteroplasmy of the mitochondrial (mt) transcriptional unit of nad3-orf156 (atp8) was studied in a nucleus-cytoplasm (NC) hybrid of Triticum timopheevi with the D plasmon from the maternal Aegilops squarrosa and compared with that of the parental lines. The tetraploid NC hybrid and the parental lines both showed varying degrees of heteroplasmy in this mtDNA region. The G plasmon of the paternal T. timopheevi possessed five sequence types, while two sequence types were detected in the D plasmon of Ae. squarrosa. The NC hybrid possessed all the five sequence types identical to those of the paternal parent in a 30% relative stochiometry. The remaining 70% comprised only one of the two maternal sequence types, suggestive of strong and selective NC interaction. No novel sequence types were detected and the relative stoichiometries of the paternal sequence types were conserved in the NC hybrid. No paternal-identical or -related sequences were detected in the maternal D plasmon. These results provide evidence of the paternal transmission of the mtDNA and possibly account for the origin of the observed mtDNA heteroplasmy in the NC hybrid.