Pines, eucalypts and cork oak are ecologically and economically important species for wood, paper and cork industries. The identification of candidate genes and understanding of transcriptional regulation mechanisms related to wood and cork formation are considered as very important in breeding and conservation programmes for these species. The assessment of high-quality RNAs from different species, genotypes and tissues is required for transcriptomics studies. Most of the available protocols of total RNA isolation are suitable for needle or leaf tissue, including that reported by Chang et al. Here, an improved protocol for total RNA extraction from secondary tissues based on the protocol by Chang et al. is presented. The total RNA samples isolated from secondary tissues of Pinus sp., Eucalyptus globulus Labill. and Quercus suber L. showed good yield and quality. In the extracted RNA, the small RNA fraction was preserved, being suitable for different downstream approaches, such as expression analysis of coding and non-coding genes. It is considered that this protocol could be useful to other researchers who are working in the transcriptomics field of woody species.