Biotechnological techniques comprise useful tools for the conservation of endangered plant genetic resources. In the present work, polymorphism and usefulness of amplified fragment length polymorphism (AFLP) markers in assessing the genetic diversity in populations of Billbergia zebrina were investigated in nodular cultures and adult plants of the species. AFLP markers revealed moderate-to-high genetic diversity based on the estimations of Nei's gene diversity (mean He = 0.28), Shannon index of diversity (mean HS = 0.48) and the number of polymorphic fragments (mean of 56.17 polymorphic fragments over six primer pairs). In comparison to published studies of population genetics performed in other bromeliad species, the present study suggests that natural populations of B. zebrina likely maintain high levels of genetic diversity, an important feature towards conservation of plant genetic resources. The results obtained reveal that AFLP markers comprise a powerful tool in order to assess the levels of genetic diversity in natural populations of this endangered species. Integrating AFLP markers with in vitro propagation techniques is understood as an adequate strategy for conservation programmes of this species.