A multiplex PCR assay with five primers targeting the 16S and 23S rRNA genes was developed for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. The selected primers amplify a 257-bp fragment from A. cryaerophilus, a 401-bp fragment from A. butzleri and a 641-bp fragment from A. skirrowii. No PCR product was generated for closely related bacteria including Campylobacter and Helicobacter species. The assay was useful to identify cultures after in vitro cultivation and to detect and identify A. butlzeri and A. cryaerophilus from poultry samples present in 24-h old enrichment in Arcobacter broth with cefoperazone, amphotericin and teicoplanin (CAT)-supplement.