Aim The microbial differences between peri‐implantitis and periodontitis in the same subjects were examined using 16S rRNA gene clone library analysis and real‐time polymerase chain reaction. Materials and methods Subgingival plaque samples were taken from the deepest pockets of peri‐implantitis and periodontitis sites in six subjects. The prevalence of bacteria was analysed using a 16S rRNA gene clone library and real‐time polymerase chain reaction. Results A total of 333 different taxa were identified from 799 sequenced clones; 231 (69%) were uncultivated phylotypes, of which 75 were novel. The numbers of bacterial taxa identified at the sites of peri‐implantitis and periodontitis were 192 and 148 respectively. The microbial composition of peri‐implantitis was more diverse when compared with that of periodontitis. Fusobacterium spp. and Streptococcus spp. were predominant in both peri‐implantitis and periodontitis, while bacteria such as Parvimonas micra were only detected in peri‐implantitis. The prevalence of periodontopathic bacteria was not high, while quantitative evaluation revealed that, in most cases, prevalence was higher at peri‐implantitis sites than at periodontitis sites. Conclusions The biofilm in peri‐implantitis showed a more complex microbial composition when compared with periodontitis. Common periodontopathic bacteria showed low prevalence, and several bacteria were identified as candidate pathogens in peri‐implantitis.