ABSTRACT Bacteria belonging to the Enterobacter genus are frequently isolated from clinical samples but are unusual causative agents of orthopedic implant infections. Twelve genetic clusters (clusters I to XII) and one sequence crowd (sequence crowd xiii ) can be distinguished within the Enterobacter cloacae nomenspecies on the basis of hsp60 sequence analysis, and until now, none of these clusters could be specifically associated with a disease. In order to investigate if specific genetic clusters would be involved in infections of orthopedic material, two series of bacterial clinical isolates identified as E. cloacae by routine phenotypic identification methods were collected either from infected orthopedic implants ( n = 21) or from randomly selected samples of diverse anatomical origins (control; n = 52). Analysis of the hsp60 gene showed that genetic clusters III, VI, and VIII were the most frequent genetic clusters detected in the control group, whereas cluster III was poorly represented among the orthopedic implant isolates ( P = 0.006). On the other hand, E. hormaechei (clusters VI and VIII), but not cluster III, is predominantly associated with infections of orthopedic implants and, more specifically, with infected material in the hip ( P = 0.019). These results support the hypothesis that, among the isolates within the E. cloacae complex, E. hormaechei and hsp60 gene sequencing-based cluster III are involved in pathogenesis in different ways and highlight the need for more accurate routine Enterobacter identification methods.