Eucalyptus nitens Maiden is a fast growing species used principally for pulpwood and solid-wood production, it is preferred over other Eucalyptus species at high elevation, due its cold tolerance. Studies show that the frost tolerance in Eucalyptus spp. is controlled by genetic factor. One tools that has been used in recent years to analyze gene expression is the transcriptomic analysis of sequences obtained by RNA-seq. There are many bioinformatics tools that allow this analysis, however, not all have the same precision. Among the most used in the detection of differentially expressed genes, are DEGseq and edgeR, so the objective was to identify by in silico analysis, genes differentially expressed during the low temperature acclimatization of E. nitens, associating these to tolerance to freezing. Three treatment conditions was used: acclimated before freezing (CABF), cold acclimated after freezing (CAAF), deacclimated (DA) and the control, non- acclimated (NA). The results obtained suggest that it is better to use the package edgeR for the analysis of differential expression, because it detects a smaller amount of false positives, it is when there are a few biological replicates and increases its fidelity when analyzing the biological variability of the samples. The NA-ADH comparison was the one with the most genes detected, while the AAH-DA was the lowest. In all comparisons we found a greater amount of negatively regulated genes than positively regulated genes. Within the differentially expressed genes with significance for the key comparisons we found proteins dehydrins and CBF transcription factors, important in the process of acclimatization at low temperature. Through the analysis of differentially expressed genes and genetic ontology LEA (Late embryogenesis abundant) genes were identified, within which EniDHN1 and EniDHN2, hydrophilic proteins and the CBF (C-repeat binding factor) transcription factor can be highlighted as genes asssociated with tolerance to low temperatures.