This study was conducted to establish a protocol for in vitro flowering of Centaurea cyanus and cell cultures of Centaurea montana. In four weeks, 50 to 60 adventitious shoots developed on leaf explants cultured in MS medium supplemented with 2.0 mg/L benzylaminopurine (BAP) and 0.1 mg/L indole-3-acetic acid (IAA). In vitro flowering and seed set occurred in C. cyanus when the shoots were incubated on MS basal medium supplemented with B5 vitamins, 100 mg/L myo-inositol and 30 g/L sucrose for 4 weeks under 16 h photoperiod. Young leaves of C. montana cultured on MS medium supplemented with 1.0 to 6.0 mg/L 2, 4-dichloroxypenoxyacetic acid (2, 4-D) alone or in combination with 0.5 mg/L BAP generated callus. Liquid MS medium containing 2.0 mg/L 2, 4-D produced greater fresh weight (FW), dry weight (DW) and packed cell volume (PCV) compared to MS medium with 1 mg/L 2, 4-D in a 30 day culture cycle. The results indicate that MS medium modified with appropriate phytohormones can be used to achieve efficient shoot regeneration and in vitro flowering of C. cyanus and cell cultures of C. montana.