Micropropagation techniques are valuable tools for propagating, conserving and restoring trees. An efficient micropropagation method involving axillary shoot proliferation of material obtained from mature European alder (Alnus glutinosa (L.) Gaertn.) trees was developed. Branch segments from trees aged 20–30 years were forced to flush, and explants derived from new shoots were cultured on Woody Plant Medium supplemented with 8.88 µM benzyladenine and 2.85µM indole-3-acetic-acid. In vitro establishment was achieved in all five genotypes evaluated. Shoot cultures were maintained by sequential subculture of explants on the same medium supplemented with 0.88–0.44 µM benzyladenine and 2.85 µM indole-3-acetic acid. Transfer to fresh medium every 3 weeks during a 9-week multiplication period and the inclusion of 2.28 µM zeatin during the last 3 weeks of culture improved the multiplication rate and shoot quality. Use of 2% glucose as the carbohydrate source produced better results than 3% sucrose for shoot proliferation. In vitro rooting of shoots was achieved with 2% glucose and 0.49 µM indole-3-butyric acid for 7 days, followed by in vitro culture on auxin-free medium for 21 days. Rooted plantlets were acclimatized to the greenhouse and were viable for reintroduction into the natural habitat.