In Staphylococcus epidermidis, ica locus encodes for the synthesis of a polysaccharide intercellular adhesin (slime or biofilm). A multiplex polymerase chain reaction (PCR) for the detection of the five individual genes of ica locus was developed, with the aim to probe the set of genes in a large collection of Staphylococcus epidermidis clinical isolates. Single representative fragments for icaR, icaA, icaD, icaB, and icaC genes were selected. Multiplex PCR was applied to two reference Staphylococcus epidermidis strains [the non-biofilm-forming ATCC 12228 and the biofilm-forming ATCC 35984 (RP62A)] and to 400 clinical isolates of Staphylococcus epidermidis from orthopedic prosthesis associated infections. The gene profile was compared with the phenotypic biofilm-forming ability, evaluated by means of an optimized Congo red agar (CRA) plate test. Among the clinical isolates, 228 (57%) turned out completely ica positive and were biofilm producing. Among the 172 non-biofilm-forming strains (43%), 164 (41%) were completely ica negative and 8 strains (2%) harbored all five ica genes. The ica locus thus proves to be a cluster of strictly linked genes, without any evidence of single gene deletion.