Classification of bacteria is mainly based on sequence comparisons of certain homologous genes such as 16S rRNA. Recently there are challenges to classify bacteria using oligonucleotide frequency pattern of nonhomologous sequences. However, the evolutionary significance of oligonucleotides longer than tetra-nucleotide is not studied well. We performed phylogenetic analysis by using the Euclidean distances calculated from the di to deca-nucleotide frequencies in bacterial genomes, and compared these oligonucleotide frequency-based tree topologies with those for 16S rRNA gene and concatenated seven genes. When oligonucleotide frequency-based trees were constructed for bacterial species with similar GC content, their topologies at genus and family level were congruent with those based on homologous genes. Our results suggest that oligonucleotide frequency is useful not only for classification of bacteria, but also for estimation of their phylogenetic relationships for closely related species.