The aim of this study was to investigate phenotypic and/or genotypic heterogeneity in coagulase-negative staphylococci (CoNS) obtained from multiple tissue samples taken perioperatively during exchange surgery from each of 19 patients with clinically and/or microbiologically proven hip prosthesis infections. CoNS are important pathogens in prosthetic hip joint infections. Several virulence factors have been suggested for CoNS, such as phenotypic variation, yet the pathogenic processes that are involved remain unclear. The PhenePlate system (PhPlate AB, Stockholm Sweden) was used for phenotyping and pulsed-field gel electrophoresis for genotyping of polymorphisms in isolates of CoNS. Furthermore, polymerase chain reaction was used to determine the presence of the icaADB gene complex in the isolates. Some patients were infected with CoNS and other species, some were infected with multiple CoNS species, although infections with Staphylococcus epidermidis alone were most common, and some were infected with different S. epidermidis clones. Phenotypic variation was found among isolates both from the same tissue sample and from different samples from the same patient, and in some cases such variation represented the presence of different clones. One-third of the patients infected with S. epidermidis carried the icaADB genes. CoNS isolates showing phenotypic and/or genotypic heterogeneity were identified in tissue samples from half of the patients. The presence of the intercellular adhesion (ica) operon does not seem to be a prerequisite for establishing infection with CoNS.