Abstract: We analyzed 10 000 expressed sequence tags (ESTs) of Eucalyptus deposited in GenBank and detected 1 775 microsatellites distributed in 1 499 EST sequences. Thus, about 15% of the EST sequences contain one or more microsatellites in the genome of Eucalyptus. Diversification of microsatellite lengths was negatively correlated with their repeat motif lengths. Apart from the triplet repeats, the abundance of the other three classes of microsatellites was also negatively correlated with their repeat motif lengths. Triplet repeats are the most abundant microsatellites in the EST sequences of Eucalyptus. The overabundance of triplet repeats might result from genetic code selection. A comparison of microsatellites in the EST sequences of Eucalyptus and the transcript sequences annotated from poplar genome sequences revealed similar variation trends in microsatellite lengths and abundance with their repeat motif lengths. However, the microsatellite content and frequency of triplet repeats were significantly lower in Eucalyptus than in poplar. This might relate to the lower expression of microsatellite-containing genes. We subsequently designed simple sequence repeat (SSR) primers and successfully detected microsatellite loci.