Campylobacter bacteria are important foodborne pathogens that cause acute diarrheal illness, and infection is often associated with contaminated poultry. In a blind observational study, the prevalence and resistance profiles of thermophilic Campylobacter strains collected from different poultry production systems were tested against the clinically used antibiotics ciprofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Campylobacter strains were isolated from chickens in rural production systems, a free-range commercial facility, and industrially raised broiler and egg-laying chickens all situated in KwaZulu-Natal, South Africa. Isolates were collected from the chicken cecae and were identified with conventional methods and tested for antibiotic resistance with the Clinical and Laboratory Standards Institute agar dilution method. The prevalence of Campylobacter spp. isolates in chickens was 68% (56 samples) in rural production, 47% (140 samples) in commercial free-range broilers, 47% (133 samples) in industrial broilers, and 94% (34 samples) in industrial layer hens. Isolates from the rurally raised chickens showed significantly (P < 0.01) less resistance against ciprofloxacin (7.9%), erythromycin (0%), and tetracycline (21.6%) than those from commercially produced chickens. Isolates from the commercially raised chickens (free range and industrial) were highly resistant to tetracycline (98.9 to 100%). The incidence of gentamicin and streptomycin resistance was 1.6 and 11.5%, respectively, in commercial free-range broilers, 1.7 and 16.4%, respectively, in industrially raised broilers, and 12.9 and 40%, respectively, in industrially raised layers. It is possible that variations among the poultry production systems, including antimicrobial usage, result in differences in antibiotic resistance profiles in Campylobacter.