Microbial colonization by Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Enterobacteriaceae, and Pseudomonas species is speculated to be a factor in the progression of Chronic Obstructive Pulmonary Disease (COPD); however, the role of bacterial infection in COPD has not been completely understood (Brusse-Keizer, 2008). Researchers studying the bacteriology of COPD patients have varying conclusions on the most prevalent pathogens present in the sputum. Only a very small percentage of all bacteria can be cultured, so advanced molecular methods are needed for the diagnosis of infections (Staley, 1985). In this study, we used metagenomics based on 16S rRNA sequences to study the sputum microflora of 19 COPD patients admitted to The Penn State Hershey Medical Center. In comparison with the 454 metagenomics sequencing, culture methods were also used, further identifying cultured organisms by individual 16S rRNA sequencing. While we identified many of the pathogens known to contribute to COPD, we also identified Actinomyces spp., Tannerella forsythia, Gemella morbillorum, Eikenella corrodens, and other opportunistic pathogens in the respiratory tracts of COPD patients. As discovered for known COPD pathogens, these species may also be contributing to disease symptoms. Metagenomic approaches have a tremendous capability to generate compositional data, and it has provided us with novel insight into the bacterial colonization of the lower airways of COPD patients.