DUOII is a multi-ovary wheat line with two or three pistils and three stamens in each floret. The multi-ovary trait of DUOII is controlled by a dominant gene, whose expression can be suppressed by the heterogeneous cytoplasm of TeZhiI (TZI), a line with the nucleus of common wheat and the cytoplasm of Aegilops. DUOII (♀) × TZI (♂) shows multi-ovary trait, while TZI (♀) × DUOII (♂) shows mono-ovary. Observing the developmental process, we found that the critical stage of additional pistil primordium development was when the young spikes were 2-6 mm long. To elucidate the molecular mechanisms that are responsible for the heterogeneous cytoplasmic suppression of the multi-ovary gene, we RNA-sequenced the entire transcriptome of 2-6 mm long young spikes obtained from the reciprocal crosses between DUOII and TZI. A total of 600 differentially expressed genes (DEGs) was identified. Functional annotation of these DEGs showed that the heterogeneous cytoplasmic suppression of additional pistil development mainly involved four pathways, i.e., chloroplast metabolism, DNA replication and repair, hormone signal transduction, and trehalose-6-phosphate in the primordium development stage, which cooperated to modulate the multi-ovary gene expression under heterogeneous cytoplasmic suppression.