Rootstock is widely used for the cultivation of citrus fruits because it brings resistance or tolerance to diseases or environmental constrains and modulates the fruit quality. Polyploidization is a widespread improvement strategy in citrus. The objective was to evaluate the effect of rootstock and ploidy level on the composition of essential oils. Two trials were conducted, one displaying a 'Navelina' orange grafted on three rootstocks and a second combining two ploidy levels (di and tetraploid) of scion ('Pineapple' orange) and rootstock ('Carrizo' citrange). The composition of peel essential oil (PEO) was analyzed by gas chromatography coupled with mass spectrometry, and a panel of experts analyzed its flavor variation with a triangle test approach. The rootstock influenced the yield and composition of the orange PEO, with a low impact on flavor. Neither the rootstock nor the scion ploidy level affected the PEO yield. Only the tetraploid level of the scion significantly modified the PEO composition, reducing the oxygenated compound fraction. Sensitive significant differences were detected between the reference sample (diploid scion–diploid rootstock) and the three other combinations. These results suggest that for the profiling of an aromatic flavor, the rootstock is a key element as is the ploidy level of the scion.