The WRKY transcription factor family is involved in multiple biological processes, especially in the transcriptional regulation associated with plant abiotic stress response. This gene family has not been previously characterized in Eucalyptus globulus, a sensitive species to freezing temperatures, able to develop a certain degree of frost tolerance when exposed to low but nonfreezing temperatures, a phenomenon known as cold acclimation. In this study, based on the RNA-seq data from cold-acclimated E. globulus plants, a total of 51 EglWRKY genes were identified. Phylogenetic analysis allowed to classify these genes into three main groups and five subgroups, according to their putative orthologous genes in Arabidopsis. The expression patterns of 51 EglWRKY genes under cold stress were determined by in silico DEG analysis in leaf and root. Transcriptome profiling indicated that the expression of multiple EglWRKY genes is activated under chilling (4 degrees C) and freezing temperatures (- 6 degrees C). Expression patterns derived from qRT-PCR showed that 11 EglWRKY genes are regulated during cold acclimation in leaf tissue. These results provide the basis for further studies and analysis of WRKY genes to identify their function and molecular mechanisms involved in plant abiotic stress responses in E. globulus.