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Background/purpose Interlaboratory comparison of pulsed-field gel electrophoresis (PFGE) patterns is difficult. A key reference of standardized PFGE protocol for Acinetobacter baumannii may address this issue. This study aimed to determine restriction enzymes with rare cutting sites on A baumannii genomes and evaluate their cost-effectiveness, discriminatory power, and interlaboratory consistence of band assignments. Methods There were 42 A baumannii isolates collected, including nine from three hospital outbreaks and 33 sporadic isolates. The numbers of cutting sites for the restriction enzymes were explored using the "Restriction Digest and PFGE" program. The cost-effectiveness for PFGE analysis was evaluated for the tested restriction enzymes, while its discriminatory ability was expressed through a discriminatory index and 95% confidence interval. The interlaboratory consistence of band assignments was evaluated for the 42 A baumannii isolates. Results ApaI was the most cost-effective restriction enzyme for a PFGE protocol for A baumannii . Both AscI and AsiSI were reasonable in terms of costs. ApaI, AscI, and AsiSI exhibited similar discriminatory indices. ApaI generated more than 40 fragments that were close and not easy to resolve, resulting in less consistence of band assignments. AscI and AsiSI generated 10–20 fragments that were clearly resolved, resulting in higher consistence of band assignments. AscI exhibited a close discriminatory power to that of AsiSI and at half of the cost of AsiSI for PFGE analysis. Conclusion We recommend AscI as the primary enzyme and AsiSI as the secondary enzyme for standardizing the PFGE protocol and interlaboratory comparisons of A baumannii .