Variety DUOII is a multi-ovary line of common wheat (Triticum aestivum L.) that has two or three pistils and three stamens. The multi-ovary trait is controlled by a dominant gene, the expression of which can be suppressed by the special heterogeneous cytoplasm of line TeZhiI (TZI). TZI has the nucleus of common wheat and the cytoplasm of Aegilops. DUOII (♀) × TZI (♂) shows the multi-ovary trait, whereas TZI (♀) × DUOII (♂) shows the mono-ovary trait. DNA methylation affects gene expression and plays a crucial role in organ and tissue differentiation. In order to study the relationship between DNA methylation and the suppression of the multi-ovary gene, we used methylation-sensitive amplification polymorphisms (MSAP) to assess the DNA methylation status of the reciprocal crosses. Genome-wide, 14 584 CCGG sites were detected and the overall methylation levels were 31.10% and 30.76% in the respective crosses DUOII × TZI and TZI × DUOII. Compared with DUOII × TZI, TZI × DUOII showed 672 sites (4.61%) in which methylation–demethylation processes occurred. The results showed that the special heterogeneous cytoplasm significantly changed DNA methylation, and this might have suppressed the multi-ovary gene. The results provide insight into the changing patterns of DNA methylation in the suppression of the multi-ovary gene, and provide essential background for further studies on the underlying mechanisms of heterogeneous cytoplasm suppression of the expression of the multi-ovary gene in wheat.