The response of wheat to elevated atmospheric CO2 concentration (e[CO2]) within a single generation is being cumulatively studied, but multigenerational effects of e[CO2] on wheat is largely unknown. In this study, the emerging spatially resolved metabolomics was used to visualize the spatial distributions of amino acids and organic acids in wheat caryopsis exposed to multigenerational e[CO2]. These results showed that nine kinds of amino acids and three kinds of organic acids exhibited embryo-distribution pattern in wheat caryopsis under sequential atmospheric CO2 concentration (a[CO2]). Additionally, e[CO2] can induce nonuniformly spatial distribution of amino acids and organic acids. Further inspection of the differential accumulation of amino acids in embryo/endosperm under multigenerational e[CO2] exposure indicated that the abundance of valine, glutamine, glutamic acid and histidine in embryo was decreased by multigenerational e[CO2] exposure, whereas abundance of lysine, aspartic acid and glutamic acid in endosperm showed different trends. Multigenerational e[CO2] exposure decreased the abundance of ascorbic acid and cis-aconitic acid, regardless of regions, while increased the abundance of malic acid in endosperm. It was suggested that the long-term e[CO2] cause inconsistent changes in embryo and endosperm, which may lead to potential impacts on human dietary health and crop offspring vigor.