Abstract: Flavonoids obtained from different tissues of Citrus sinensis ‘Cara Cara’ and ‘Seike’ were analyzed by high performance liquid chromatography tandem quadrupole and time of flight mass spectrometry (HPLC-Q-TOF/MS) in positive ion monitoring mode. Ten flavonoids were identified with authentic standards, while three more (4',5,7,8-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone and 3,5,6,7,8,3',4'-heptamethoxyflavone) were annotated based on exact m/z data, MS2 fragmentation pattern and elution order described in the literature. Analysis of the relative content of the flavonoids (according to their average peak areas) showed that flavedo accumulated the highest levels of flavonoids in different tissues of navel oranges while the lowest accumulation was obtained in juice veciles. Sinensetin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone and 4',5,6,7-tetramethoxyflavone were found to be the major polymethoxyflavones (PMFS) in flavedo and hesperidin and naringin were the main flavonoids in albedo, segment membranes and juice vesicles of navel oranges. Relative content of flavonoids in albedo, segment membranes and juice vesicles showed no significant differences between the two cultivars, except for the levels of hesperidin, 3,5,6,7,3',4'-hexamethoxyflavone and tangeretin. These results lay a foundation for further investigation and usage of the bioactive compounds in navel oranges.