Background: Recent studies have suggested that changes in the prevalence and/or proportion of distinct microorganisms characterize the subgingival microbial profiles of populations around the world. At present, no information is available on the subgingival microbiota of Mexican subjects. The purpose of the present study was to determine the microbial composition of subgingival plaque in Mexican subjects with untreated chronic periodontitis. Methods: A total of 44 chronic periodontitis and 20 periodontally healthy subjects (who were currently non-smokers) were selected. Clinical measurements including plaque accumulation, gingival erythema, bleeding on probing, suppuration, probing depth, and attachment level were recorded at six sites of every tooth. Up to 28 subgingival plaque samples were obtained from each subject and individually analyzed to determine the levels, proportion, and prevalence of 40 microbial species using the checkerboard DNA-DNA hybridization technique. Results: Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythensis were the only species that presented higher mean levels in periodontitis subjects. The proportions of P. gingivalis (P < 0.001), T. forsythensis (P < 0.01), and red complex species (P. gingivalis, T. forsythensis, and T. denticola; P < 0.001) as a group were also significantly higher in periodontitis subjects. Periodontally healthy subjects harbored a significantly larger proportion of Actinomyces species (P < 0.05). No significant differences were detected in the percentage of carriers of any of the species tested. Conclusions: Our results revealed that the subgingival microbiota of untreated chronic periodontitis Mexican subjects was characterized by increases in the level, prevalence, and proportion of classic periodontal pathogens. However, the prevalence and proportion of specific microbial species varied significantly from the results of other reports on subjects from different geographical locations.