Eucalypt species are among the most widely planted genus of woody angiosperms in the world with a global plantation estate of 18 million hectares. The major commercial species, have fast growth rates, excellent form, wood and fiber properties for pulp and paper, and are potential stocks for biofuel and bioenergy. Eucalypts are in early stages of domestication and are therefore attractive targets for biotechnologically-led improvement. The DOE-JGI Eucalyptus Genome Project has resulted in an annotated genome assembly for Eucalyptus grandis, providing a tool for molecular and genetic understanding of eucalypts. To complement this effort, we present our work to establish a reference genome sequence and gene catalogue for Eucalyptus globulus, a major temperate pulpwood plantation species internationally and a target for tree improvement in a number of countries. We generated 270 Million Illumina HiSeq paired reads from the DNA of E. globulus clone X46. Among the 195 M high-quality reads, 2.6% are chloroplast and 95% of the remaining ones map to the E. grandis assembly, enabling us to build a reference-guided genome assembly of E. globulus. To create a gene catalogue for E. globulus, we generated 200 Million HiSeq reads from RNA of five tissues of X46 representing well watered and drought conditions. From the 151 M high-quality reads, 96% mapped to the genome sequence. We compared our catalogue to the E.grandis gene annotation, thereby supporting 83% of the E. grandis annotations with E. globulus RNA-Seq reads, as well as identifying novel transcripts uniquely documented by E. globulus sequences.