Abstract Objective: To evaluate changes in subgingival microbiota before and during the leveling and alignment orthodontic stage using the polymerase chain reaction (PCR) method. Materials and Methods: Thirty orthodontic patients (17 females and 13 males; aged 16.7 ± 6.5 y) were included in this study. Subgingival microbial samples were taken from the disto-buccal gingival crevice of the left upper central incisors, the left lower central incisors, the mesio-buccal gingival crevice of the left upper first molars, and the left lower first molars, at four different times: at baseline, before placement of orthodontic appliances (T1), and 1 week (T2), 3 months (T3), and 6 months after placement of orthodontic appliances (T4). DNA was extracted from the samples, and the 16S rRNA-based PCR detection method was used to determine the prevalence of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobactor rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, and Treponema denticola, which are considered as putative periodontopathogens. Results: The frequency of T forsythia, C rectus, and P nigrescens significantly increased after placement of orthodontic appliances. For the other species, the frequency tended to increase but no statistically significant difference was noted. The frequency of the change, representing microorganisms not existing at T1 but newly developing at T2, T3, and T4, was higher at the molars than at the incisors. Conclusion: The placement of orthodontic appliances affects the subgingival microbial composition even during the early period of orthodontic treatment, increasing the prevalence of periodontopathogens, especially in the molar region.