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Background  Micro RNAs are a class of small non coding RNAs of 20–24 nucleotides transcribed as single stranded precursors from MIR gene loci. Initially described as post-transcriptional regulators involved in development, two decades ago, miRNAs have been proven to regulate a wide range of processes in plants such as germination, morphology and responses to biotic and abiotic stress. Despite wide conservation in plants, a number of miRNAs are lineage specific. We describe the first genome wide survey of  Eucalyptus  miRNAs based on high throughput sequencing.    Results  In addition to discovering small RNA sequences, MIR loci were mapped onto the reference genome and interspecific variability investigated. Sequencing was carried out for the two most world widely planted species,  E. grandis  and  E. globulus.  To maximize discovery,  E. grandis  samples were from BRASUZ1, the same tree whose genome provided the reference sequence. Interspecific analysis reinforces the variability in small RNA repertoire even between closely related species. Characterization of  Eucalyptus  small RNA sequences showed 95 orthologous to conserved miRNAs and 193 novel miRNAs.  In silico  target prediction confirmed 163 novel miRNAs and degradome sequencing experimentally confirmed several hundred targets. Experimental evidence based on the exclusive expression of a set of small RNAs across 16 species within Myrtaceae further highlighted variable patterns of conservation and diversity of these regulatory elements.    Conclusions  The description of miRNAs in  Eucalyptus  contributes to scientific knowledge of this vast genre, which is the most widely planted hardwood crop in the tropical and subtropical world, adding another important element to the annotation of  Eucalyptus grandis  reference genome.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-015-2322-6) contains supplementary material, which is available to authorized users.

Genome-wide discovery and validation of Eucalyptus small RNAs reveals variable patterns of conservation and diversity across species of Myrtaceae