The basic-helix-loop-helix (bHLH) transcription factors superfamily is present in eukaryotes and has been widely characterized in several species. The study of these proteins has focused on processes involved in plant growth and development. However, it has also been shown that bHLH plays a relevant role in processes of response to abiotic stress. In this study, the bHLH family in Eucalyptus was systematically characterized for the first time, using the Eucalyptus grandis genome. 153 bHLH genes in the E. grandis genome were identified, which were characterized phylogenetically and structurally. Then, a cold-tolerant hybrid of Eucalyptus (Eucalyptus nitens x Eucalyptus globulus) to generate a reference assembly was used to study the alternative splicing events produced in the cold response. 44 bHLH candidate genes to generate alternative splicing events in the hybrid were identified. These genes had exons differentially expressed in cold acclimation conditions. The splicing event prediction was further validated by qRT-PCR in three bHLH genes. Through this experiment was possible to determine that two of them were differentially expressed, so that new isoforms of these genes were being produced under cold acclimation conditions.