ObjectivesThe aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments.ResultsWe obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140mg/L, allowed the maximum isoflavonoid production in plantlets (11.9mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9mg/g dry weight) was obtained using sucrose 60g/L, for 3.5days.ConclusionThis work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.