In this study, the flowers of Forsythia × intermedia, Forsythia suspensa and Forsythia ovata are presented as valued raw materials for isolation of rutin, an industrial and pharmacologically active flavonoid. An efficient strategy based on three high-performance chromatographic techniques was established to screen and purify the target compound from the plant matrices. In the first step, an UHPLC-PDA-ESI-MS3 assay documented the relatively simple phytochemical profiles of the flower extracts and led to the identification of ten polyphenols classified as caffeoyl acid derivatives, flavonoids, phenylethanoids and lignans, with rutin as the major constituent. Significant levels of rutin (20.0–57.6mg/g dw) were found in the flowers by the validated HPLC-PDA method, with the highest values observed for F. suspensa. Finally, a novel, fast, single-step HPCCC method was developed and successfully applied for the separation of rutin from the plant samples. The ethyl acetate-n-butanol-water (4:1:5, v/v/v) was selected as the optimum two-phase solvent system for both analytical and preparative normal (NP) and reversed-phase (RP) HPCCC separations. Under optimal RP-HPCCC conditions, 155 mg of the crude methanol extract was separated in a single 50min run, yielding 14.6mg of rutin with 97.2% HPLC purity and 95.9% recovery. The process efficiency of the HPCCC rutin isolation was demonstrated to be superior to the existing HSCCC methods. The results indicated the usefulness of Forsythia flowers and the developed RP-HPCCC methodology for practical and commercial applications.