The purpose of this study is to identify the presence of Campylobacter species (C. rectus, C. concisus, C. gracilis and C. ureolyticus) in Chilean patients with chronic periodontitis, and to establish the relationship of these microorganisms with the periodontal conditions of smoker and non-smoker patients. Subgingival plaque samples were collected from four periodontal-affected tissue sites of 15 smoker and 15 non-smoker patients with chronic periodontitis. A sample per quadrant was obtained, with a probing depth of ≥6 mm and an insertion loss of >3 mm in each patient. Polymerase chain reaction (PCR) was performed with the specific 16S rDNA primers for the molecular detection of C. rectus and C. gracilis, specific cpn60 primers for C. concisus and hsp60 primers gene for C. ureolyticus. Campylobacter species showed an overall prevalence of 93.3% in periodontal patients, while C. rectus was the most frequent one (80%), followed by C. concisus (66.7%), C. gracilis (33.3%), and C. ureolyticus (10%). Only C. gracilis showed a statistically significant (p = 0.002) association with chronic periodontitis among samples from smoker and non-smoker patients. A high prevalence of the Campylobacter genus in the analyzed populations (93.3%) was found, being C. rectus the most frequent (80%) species. Besides, C. gracilis showed a statistically significant association between smoker state and chronic periodontitis.