Globally the demand for forestry resources is booming, and consequently innovative approaches to cultivate valuable hardwoods, such as eucalypts, are crucial. Although micropropagation offers a means to clonally propagate desirable cultivars, this technique is often variety-specific for eucalypts. Thus, recalcitrant genotypes are often excluded from micropropagation programmes as development requires large investments of time, money and expertise. In this study, a minimal maintenance medium was developed to reduce subculturing frequency (∼8 days) and to evaluate each micropropagation stage independently. Furthermore, a generic protocol for use across different varieties was developed by assessing the in vitro performance of three different Eucalyptus grandis x Eucalyptus nitens varieties (varieties 1–3) across various treatments. The purpose of this protocol was to ease the in vitro establishment of novel eucalypt varieties by providing a foundation for future protocol optimisation. During multiplication, medium containing 0.5 mg l−1meta-topolin with 0.1 mg l−1 of indole acetic acid (IAA) induced the highest bud proliferation and shoot elongation for varieties 1 (59.1 ± 2.9; 1.2 cm ± 0.08), 2 (62.3 ± 2.7; 1.5 cm ± 0.09) and 3 (58.1 ± 3; 1.2 cm ± 0.07). Following multiplication, the rooting treatment that achieved the most consistent rooting percentages among varieties 1 (33.4%), 2 (43.5%) and 3 (34.3%) incorporated 0.029 mg l−1 of racemic-GR24 with 0.5 mg l−1 of IAA. Additionally, root vigour was assessed by measuring the root number and length, which varied considerably based on the variety and treatment in question. Overall, variety 2 was the most amenable to plant growth regulators and upon further investigation was found to possess equal expression levels of the auxin transporters PIN1 and AUX1. Conversely, the other varieties displayed unequal ratios of these transporters. Considering that auxins are principal media components, these expression profiles may serve as markers to identify eucalypt cultivars amenable to micropropagation. [ABSTRACT FROM AUTHOR]