Flower color is the major characteristics and critical breeding program for most Rhododendron species.However, little is known about their coloration mechanism and color inheritance. In this study, petal pigmentconstituents of three Rhododendron pulchrum Sweet cultivars with different colors were clarified based on LCESI-MS/MS method. Using a broad-targeted metabolomic approach, a total of 149 flavonoids and theirglycosylated or methylated derivatives were identified, including 18 anthocyanins (Pg, Cy, Dp, Pn, Pt, and Mv)and 32 flavonols (mainly kaempferol 3-O-glycosides and quercetin 3-O-glycosides). Moreover, anthocyaninswere mainly represented by anthocyanidin-3-O-glycosides (glucoside, rutinoside, galactoside, and di-glycosides).Flavone and C-glycosylated flavone were major second metabolites responsible for the differenceamong three different R. pulchrum cultivars. The accumulation of total flavonoids displayed a clear phenotypicvariation: cultivars ‘zihe’ and ‘fenhe’ were clustered together, while ‘baihe’ was clustered alone in the HCAanalysis. The composition and content of anthocyanins were more complex in colored flowers (‘zehe’ and

‘fenhe’) than in white flower (‘baihe’). This study further enhanced our understanding on the flavonoids profileof flower coloration and will provide biochemical basis for further genetic breeding in Rhododendron species.