Stem segments from apical shoot tips of Polygala myrtifolia were used as primary explants to establish in vitro cultures. Axillary shoots produced on non-contaminated explants were excised and recultured in the same medium to increase the stock of shoot cultures. Equal molar concentrations of five cytokinins [2-isopentenyladenine, kinetin, zeatin, N⁶-benzyladenine (BA), and adenine] were tested for ability to induce axillary shoot development from double-node stem segments. The highest rate of axillary shoot proliferation was induced on Murashige and Skoog agar medium supplemented with 1.8 μM BA. Seven indole-3-acetic acid (IAA) concentrations (0, 2.9, 5.7, 8.6, 11.4, 14.3, 17.1 μM) were tested to determine the optimum conditions for in vitro rooting of microshoots. Up to 72% of the microshoots rooted with 14.3 μM IAA. Other auxins tested, α-naphthaleneacetic acid and indole-3-butyric acid, were less effective than IAA in inducing adventitious root formation. All rooted plantlets having more than three roots were successfully established in soil.