Species of Eucalyptus are keystone species for ecological studies in their natural ranges and are extensively planted in the tropical and subtropical regions of the world to supply high-quality woody biomass for various applications. We report the development of a selected set of 20 dinucleotide and trinucleotide repeat microsatellites derived from Eucalyptus expressed sequence tags (ESTs). These microsatellites were selected for full transferability and homogeneous rate of polymorphism across species. They were evaluated for individual fingerprinting, parentage testing, and intraspecific population structure analyses in 6 of the most extensively studied and planted species worldwide, representing key phylogenetic sections of the largest subgenus Symphyomyrtus. This set of markers provides exceptional resolution for population genetics and molecular breeding applications in the genus Eucalyptus. As they were developed from conserved transcribed regions, the transferability and polymorphism of these microsatellites will most likely extend to the other 300 or more species within the same subgenus.