Background: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression.Results: We characterized the population of miRNAs of Eucalyptus grandis and compared the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined.Conclusions: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability. © 2014 Levy et al.; licensee BioMed Central Ltd.