Periodontal disease is a polymicrobial disease with a complex etiology. Many studies use the 16s rRNA gene as a molecular marker for bacterial identification associated with oral biofilm, known as dental plaque. However, the high level of conservation in the 16s rRNA gene does not provide enough resolution to discriminate between closely related bacterial species. Recent studies have successfully used the RNA polymerase subunit (rpoB) gene as an alternative molecular marker to help resolve this issue. Although there are many studies that use the rpoB gene in bacterial identification, it is usually to describe specific groups, such as the family Pasteurellaceae or the genus Veillonella. In this study, we investigated the use of the rpoB gene as an alternative molecular marker to differentiate between closely related species in the microbial community of dental plaque. To analyze bacterial diversity, genomic DNA was extracted from 28 samples, and sequenced using 454 pyrosequencing. Eight out of a total of 17 identified genera were selected for phylogenetic analyses. The 16s rRNA gene sequences from these eight genera were used to compare to the rpoB gene sequences. Neisseria and Granulicatella showed greater diversity using the 16s rRNA gene. However, there was more diversity in the genera Pseudomonas, Lautropia, Eikenella, and Haemophilus using the rpoB gene. In Cardiobacterium and Aggregatibacter, there were no differences in diversity revealed with the rpoB or the 16s rRNA gene. The use of the rpoB gene to identify and differentiate between closely related bacterial species can provide insight into organisms that may be associated with health and disease states.