The multi-ovary trait of wheat inbred line DUOII is controlled by a dominant gene whose expression can be suppressed by the heterogeneous cytoplasm of TeZhiI (TZI), another inbred line with the nucleus of common wheat and the cytoplasm of Aegilops. DUOII (female) x TZI (male) shows multi-ovary trait, while TZI (female) x DUOII (male) shows mono-ovary. To elucidate the molecular mechanism regulating heterogeneous cytoplasmic suppression of the multi-ovary gene, we performed an untargeted metabolomic analysis of 2-6 mm young spikes of reciprocal crosses between DUOII and TZI at the critical stage of additional pistil primordium development. We identified 198 annotated differentially expressed metabolites and analyzed them according to their biological functions. The results showed that these metabolites had obvious functional pathways mainly implicated in amino acid, carbohydrate, nicotinate and nicotinamide, and purine metabolism and isoquinoline alkaloid biosynthesis. We also found that shikimate, phosphoglycolic acid, nicotinamide, guanine, and xanthine might play essential roles in cytoplasmic suppression of multi-ovary trait. Chloroplast metabolism was also implicated in the nuclear-cytoplasmic effect of the multi-ovary gene. The findings provide solid theoretical and empirical foundations for future studies elucidating the mechanisms controlling heterogeneous cytoplasmic suppression of the nuclear multi-ovary gene in wheat.