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The objective was to evaluate using unstimulated saliva in detecting Aggregatibacter actinomycetemcomitans and to compare the saliva and subgingival and mucosa membrane occurrence of this periodontal pathogen in patients diagnosed with advanced periodontitis. Patients with advanced forms of periodontitis (n = 220; mean age: 54.03 ± 03 years) at stage III/IV were sampled. Unstimulated saliva, buccal cheek mucosa, and pooled subgingival plaque samples were collected. The identification of A. actinomycetemcomitans was performed using qPCR. A descriptive analysis and Wilcoxon test and analysis of variance were performed. A. actinomycetemcomitans was isolated from 28.18% of the subjects. A total of 660 samples were obtained, 220 from unstimulated saliva, 220 from buccal cheek mucosa surfaces, and 220 from pooled subgingival plaque samples. A. actinomycetemcomitans was isolated from 21.80% of unstimulated saliva, 19.50% of buccal cheek swabs, and17.70% of subgingival samples. There was a statistically significant difference between the presenceof A. actinomycetemcomitans in the unstimulated saliva samples and in the buccal cheek mucosa swab samples and pooled subgingival plaque samples (p < 0.001). These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque/buccal cheek mucosa samples and its use is adequate in the oral detection of A. actinomycetemcomitans in a cohort of patients with stage III and IV periodontitis.