A PCR-DNA enzyme immunoassay (PCR-DEIA) was developed for identification of the coagulase-positive species Staphylococcus intermedius. Two PCR primers and a hybridization probe were designed to target specific sequences of the S. intermedius thermonuclease (nuc) gene. In addition to S. intermedius reference strains, the PCR-DEIA was tested using 295 veterinary and human S. intermedius isolates. A specific 933-bp DNA fragment was successfully amplified in 281 (94.9%) S. intermedius isolates. Five canine isolates showed an unexpected 2.8-kbp band. Except for 10 amplicons derived from equine, camel, and pigeon isolates, all positive PCR results (n = 288, 96.6%) were confirmed by the colorimetric microtiter plate DEIA hybridization. Isolates that failed both in amplification and DEIA hybridization were only observed in equine isolates (10/23, 43.5%). Except for the limitations with isolates of hoofed animals, the S. intermediusnuc PCR assay has potential for rapid identification of S. intermedius and differentiation from other coagulase-positive staphylococci including S. aureus.