This study aims to evaluate the origin- and topophysical- effects of the initial explants excised from crown branches on the in vitro clonal production of mature birch. Seven culture lines were successfully established in vitro from single buds and nodes taken from top crown branches and epicormic shoots of a monumental mature white birch tree (Betula pubescens ssp. celtiberica). Murashige and Skoog medium supplemented with meta-topoline was used for shoot proliferation. The effects of explant type (apical or basal segment), vessel type and subculture period during the proliferation stage were also investigated. Results indicated that the number of shoots per explant and the height of the tallest shoot were influenced by the type of explant. Basal nodal segments performed better than apical explants and their morphogenetic ability was independent of the vessel type. Proliferation rates of shoot lines were influenced by the position of the initial shoots on the explant sources. Without considering the position of the initial explant, there were no differences in shoot proliferation between lines established from two different origins. Annual new shoots developed on upper branches and epicormic shoots originated on lower crown branches. Differences related to the ontogenetic aging between the two explant sources were not reflected in the proliferation ability of crown-derived lines. The highest proliferation rates were achieved in two lines (C3 and E7-n2), each of them established from different explant sources (upper branches versus lower branches). In epicormic shoots-derived lines, the highest number of shoots was produced in line E-7n2, which was initiated from the nodal explant closest to the apical meristem. These results indicated that clonal production of mature birch trees was influenced by the position of the initial explants on the mother shoot/branch as well as by the physiological stage of the explant source. A sixweek proliferation period improved the proliferation and rooting rates. After four weeks of culture, adventitious roots were developed spontaneously in shoots maintained in the multiplication medium. Spontaneous rooting ranged from 87 to 100% depending on the culture line. Auxin induction of adventitious roots improved the root system of rooted shoots.