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Introduction To combine Multiple Displacement Amplification (MDA) and checkerboard DNA–DNA hybridization to qualitatively and quantitatively evaluate the microbiota present in infections refractory to endodontic treatment. Methods The subjects of this study were 40 patients presenting periapical lesions refractory to endodontic treatment. Samples were taken by scraping or filing root canal walls with a #10 K-type hand file. Sample DNA was amplified by MDA, and the levels of 107 bacterial taxa were analyzed by checkerboard DNA–DNA hybridization. The taxa were divided into three distinct microbial populations, depending on their mean proportion in samples (% DNA probe counts ± SEM), as follows: dominant (≥3.0%), sub-dominant (>1.6 to 3.0%) and residual (≤1.6%) populations. The significance of differences was determined using the Mann-Whitney test. Results The taxa present with the highest mean proportions (constituting the dominant population) were Corynebacterium diphtheriae (8.03±0.98), Porphyromonas gingivalis (5.42±2.09), Streptococcus sobrinus (5.33±0.69), and Stenotrophomonas maltophilia (4.72±1.73). Among the sub-dominant population were Eubacterium saphenum (3.85±1.06), Helicobacter pylori (3.16±0.62), Dialister pneumosintes (3.12±1.1), Clostridium difficile (2.74±0.41), Enterobacter agglomerans (2.64±0.54), Salmonella enterica (2.51±0.52), Mobiluncus mulieris (2.44±0.6), and Klebsiella oxytoca (2.32±0.66). In the population of bacteria present at the lowest mean proportions (the residual population), Bacteroides ureolyticus (0.04±0.01), Haemophilus influenzae (0.04±0.02), and Prevotella oris (0.01±0.01) were found at the lowest mean proportions. Enterococcus faecalis was detected in the residual population (0.52±0.26). Conclusion The microbial climax community in teeth refractory to endodontic treatment not only harbor medically important species, but also contains distinct microbial consortia present with different population levels.