Aim Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent.cloacae genetic cluster III which are relatively frequently encountered in clinical material. Methods and Results The molecular assays developed in this study are qPCR technology based and served to identify both Ent.hormaechei and Ent.cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent.hormaechei subsp. steigerwaltii and Ent.cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P<0 center dot 05). Conclusion The qPCR assays detecting Ent.hormaechei and Ent.cloacae genetic cluster III demonstrated high sensitivity and specificity. Significance and Impact of the Study The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent.cloacae complex. The improved identifications obtained can specifically assist analysis of Ent.hormaechei and Ent.cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent.cloacae cluster III and systemic infection that deserves further attention.