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The genus Eucalyptus encompasses several species of industrial importance. Many of these species have been subjected to genetic characterization using different kinds of DNA markers. More than 1000 microsatellites have been identified from the genome of eucalypts and they are highly amenable for cross species transferability. During cross amplification of microsatellites, homoplasy is reported in many species in which although the allele size might be the same, the sequences are not. Thus, it is essential to ascertain the DNA sequence homology with source and target microsatellite repeats. Accordingly, fifty five alleles from six microsatellite loci (ECc1, ECc2, Eg61, Embra100, Embra1468 and Embra2002) were amplified in two inter-specifi c hybrid populations (Eucalyptus tereticornis × E. grandis and E. tereticornis × E. camaldulensis) and sequenced. The results showed that all the microsatellite loci were amplifying the target repeat types except for the loci Eg61 and Embra2002. The locus Eg61 has target repeat of (CAA)(GAT) but the sampled alleles had either (CAA)(GAT) or (GAT) alone. Similarly, the Embra2002 locus was targeting interrupting repeats of (CCA)..(CCA), but the sequenced alleles had repeats of (CCA) with or without interruption. Nevertheless, the allele size estimated in electrophoresis for hybrids was in conformity with that of the parent alleles. This study suggests the need for validation of the repeat characteristics of microsatellites by sequencing of the alleles particularly in cross species amplification.