Introduction: Campylobacter is a major cause of gastroenteritis in developed countries. In Brazil, the incidence of this disease and consequently those that may occur after the infection is unknown. The prevalence of Campylobacter in raw poultry products varies widely. Although Brazil is the largest world exporter of chicken there is little information on the contamination of this product by Campylobacter. Objective: develop a multiplex PCR for the detection and differentiation between C. jejuni and C coli. Methods: The PCR was developed using mapA primers specific for detection of C. jejuni and ceuE primers specific for detection of C. coli which produce specific DNA fragments of 202-bp and 889-pb, respectively. The standardized PCR was tested on 11 different isolates of Campylobacter and on 22 non-Campylobacter species and the specificity was 100%. Results: Campylobacter was detected from chicken skin artificially contaminated with approximately 50 colony forming unit (CFU) of Campylobacter per 10-g, after 48 h of selective enrichment. Campylobacter spp. was detected in thirteen (46.4%) of 28 analyzed chicken carcasses purchased from local retail market. Specific DNA fragments of the PCR confirmed the presence of C. jejuni and C. coli. Conclusion: The standardized multiplex PCR was found to be sensitive and specific on the detection and simultaneously differentiation of C. jejuni and C. coli after 48h of analysis.