ABSTRACT We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo . All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro .