About 95% of Eucalyptus species present an organ known as a lignotuber, a basal woody swelling that holds a large number of dormant buds in a protected position along with carbohydrates and other nutrients. The importance of this trait in Eucalyptus species relates to its regenerative capacity, particularly in the context of coppicing practices and survival in regions of high abiotic stress, especially fire. In this study, we identified and characterized a genomic region associated with the lignotuber trait in commercially important Eucalyptus species by developing a polymorphic marker that co-segregates with lignotuber presence. The marker was then converted into a SCAR (Sequence Characterized Amplified Region) marker, validated in four other Eucalyptus species and hybrids and analyzed in silico. Our investigation presents a marker (ELig) that is effective in identifying individuals with lignotuber. In silico and Southern blot analyses show that the marker is present in a single copy region and is related to auxilin/cyclin-G associated kinase, containing a DnaJ domain. The ELig marker is an important tool that can be used to manage crosses in Eucalyptus breeding programs and inform studies involving lignotuber development and genetics.