Acinetobacter baumannii is an opportunistic pathogen of intensive care unit (ICU) patients. A. baumannii colonizes many parts of the body including the gastrointestinal tract. Endemic and epidemic strains are polyclonal. There is no clarity on the origin of polyclonality of A. baumannii. The objective of the study was to define the genetic relatedness of serial isolates and the origin of polyclonality. Serial rectal isolates from ICU patients whose rectum was colonized on �5 sampling occasions were selected. From a total of 32 eligible colonized patients, isolates from a subgroup of 13 patients (a total of 108 isolates) showing different patterns of colonization as revealed by pulsed-field gel electrophoresis (PFGE) were studied. The isolates were analyzed by PFGE pulsotypes, sequence types (STs) by multi-locus sequence typing (MLST) and clonal complex (CC) by eBURST analysis. Serial isolates constituted a mixture of identical, related and unrelated pulsotypes. Analysis by STs and CCs were less discriminatory. The data suggest a combination of an initial colonizing isolate undergoing mutation as well as colonization by independent isolates. Further clarity on the origin of diversity should be better obtained by whole-genome sequencing.