Multidrug-resistant isolates of Acinetobacter baumannii from New York City generally belong to one of three ribotypes. To assess the accuracy of ribotyping, the relatedness of representative isolates was further assessed by rep-PCR, pulsed-field gel electrophoresis (PFGE), and DNA sequencing of five genes potentially associated with antimicrobial resistance (ampC, ompA, adeB, adeR, and abeM). The isolates fell into several major groups. The first group shared the same ribotype and had common mutations affecting OmpA, AdeR, and AbeM, but consisted of two subtypes with distinctive rep-PCR and PFGE patterns and ampC mutations. The second and third groups shared common alterations in OmpA, AdeR, and AbeM, but had distinct ribotype, rep-PCR, and PFGE patterns. The resistant isolates were unrelated to the β-lactam susceptible isolates. Many of the resistant strains shared OmpA and AdeB patterns observed in several European clonal groups. Further development of a multilocus sequencing typing scheme will help determine if multidrug-resistant isolates from diverse geographic areas are indeed ancestrally related.