Humulus lupulus is a valuable medicinal plant that grows wild in forest areas of northern Iran. In recent decades with extension of village boundaries toward forest, hop habitat is suffering destruction. Optimization of hop micropropagation by shoot tip and nodal segments was conducted towards conservation of Iranian hop germplasm. Nodal segments were provided from the rootstock and after disinfection were established in the MS medium. After six weeks, explants grown in in vitro conditions were isolated, then were incubated in MS medium supplemented with various concentrations of plant growth regulators, including BA (0, 0.1, 0.5, 1 and 2 mg L-1), Kin (0, 0.1, 0.5, 1 and 2 mg L-1) and TDZ (0, 0.1, 0.5, 1 and 2 mg L-1) alone and in combination with IAA. The combination of 1 mg L-1 BA with 0.1 mg L-1 IAA was the best treatment applied in all traits evaluated, followed by 5.0 mg L-1 Kin and TDZ with 0.1 mg L-1 IAA. The best treatments for shoot tip explant proliferation were the best treatments for nodal segments proliferation, except in the case of Kin, where the best results for nodal segments proliferation was observed at a concentration of 1 mg L-1 Kin in combination with 0.1 mg L-1 IAA. For shoot rooting, the shoots were grown in vitro; and plantlets were transferred to a rooting medium containing MS medium supplemented with different concentrations IAA (0, 0.5 and 1 mg L-1) and BA (0, 0.5 and 1 mg L-1). The highest percentage of rooting was observed in half strength MS supplemented with 1 mg L-1 IAA in combination with 1 mg L-1 BA. Thus we recommend MS medium containing 1 mg L-1 IAA and 1 mg L-1 BA as a suitable micropropagation medium for industrial purposes.