The subgingival microbiome is largely uncultivated, and therefore, cultivation-based and targeted molecular approaches have limited value in examining the effect of smoking on this community. We tested the hypothesis that the subgingival biofilm is compositionally different in current and never-smokers by using an open-ended molecular approach for bacterial identification. Subgingival plaque from deep sites of current and never-smokers matched for disease was analyzed by 16S sequencing. Smokers demonstrated greater abundance of Parvimonas, Fusobacterium, Campylobacter, Bacteroides, and Treponema and lower levels of Veillonella, Neisseria, and Streptococcus. Several uncultivated Peptostreptococci, Parvimonas micra, Campylobacter gracilis, Treponema socranskii, Dialister pneumosintes, and Tannerella forsythia were elevated in this group, while Veillonella sp. oral clone B2, Neisseria sp. oral clone 2.24, Streptococcus sanguinis, and Capnocytophaga sp. clone AH015 were at lower levels. The microbial profile of smoking-associated periodontitis is distinct from that of non-smokers, with significant differences in the prevalence and abundance of disease-associated and health-compatible organisms.