The polyphenol profile of an interspecific allotetraploid somatic hybrid, achieved by protoplast fusion, the 'Valencia' sweet orange ( Citrus sinensis L. Osbeck) + 'Femminello' lemon ( Citrus limon L. Burm), and three sexual hybrids obtained by backcrosses between Femminello lemon and the allotetraploid somatic hybrid ('Valencia' + 'Femminello') was studied by liquid chromatography-ultraviolet-diode array detector-mass spectrometry (LC-UV-DAD-MS). The aim of the work was to evaluate whether superior traits and improved performance can be observed in these new genotypes. Ten flavonoids (TF), comprising seven flavanones and three flavones, and four hydroxycinnamic acids (HCA) have been characterized and quantified in all hybrids and compared with those of the respective parents. The 'Valencia' + 'Femminello' somatic hybrid shows an intermediate polyphenol composition with respect to those of the parents, with a slight prevalence of lemon influence. The three sexual hybrids show, instead, different and more complex chromatographic profiles.