A sensitive HPLC-DAD-ESI-MS/MS method was established to screen and identify the polymethoxylated flavonoids (PMFs) in the peels of 'Shatangju' mandarin ( Citrus reticulata Blanco). Eight PMF standards, including four polymethoxylated flavones, two polymethoxylated flavanones, and two polymethoxylated chalcones, were first to be analyzed in positive mode by CID-MS/MS. On the basis of the ESI-MS(n) characteristics of PMFs and the results of EIC-MS/MS experiment, 32 PMFs including 24 flavones and 8 flavanones or chalcones were screened from the complex extract of the peels of 'Shatangju' mandarin. Among them, 10 PMFs were hydroxylated polymethoxyflavonoids (OH-PMFs), and the rest were all permethoxylated PMFs. This was the first systematic report of the presence of PMFs in the peels of 'Shatangju' mandarin, especially for polymethoxylated flavanones and chalcones. Meanwhile, the contents of the three main PMFs and total flavonoids in the peels of 'Shatangju' were determined by HPLC and UV spectrophotometry, respectively. The results indicated that the developed analytical method could be employed as an effective technique for the characterization of PMFs.