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Kava (Piper methysticum) is increasingly traded internationally and there is need for a rapid method to analyze kava raw material before export. The objectives of the present study were: (i) to develop a simple and robust protocol for high throughput simultaneous quantification of kavalactones (KLs) and flavokavins (FKs) in kava and (ii) to assess its potential for quality control. Methysticin; dihydromethysticin; kavain; desmethoxyyangonin; dihydrokavain; yangonin; and flavokavin A, B and C were quantified using HPTLC in acetonic extracts of 174 kava varieties. UHPLC analysis was conducted on a subset of six varieties representing the genetic variation of the species. The genetically distinct groups of nobles, two-day and wichmannii varieties were clearly differentiated and multivariate analyses of UHPLC and HPTLC data were congruent. Noble varieties have significantly low FKs/KLs (0.13) and high kavain/flavokavin B (K/FKB = 7.31). Two-day and wichmannii varieties are characterized by high FKs/KLs (0.36, 0.21) and low K/FKB (1.5, 1.7). A high-throughput HPTLC protocol was developed with a total analytical time of 50 min for 20 samples and only 10 mL of mobile phase. The use of acetone, sonication and two different detection wavelengths improves the accuracy compared to previous HPLC studies and confirms that kava varieties exhibit distinct chemotypes clearly differentiated by their FKs/KLs profiles. These results will strengthen the use of Codex Alimentarius regional standards.