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Background Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between clinical versus vaccine strains. In the current study, the genetic profiles of clinical isolates and vaccine strains of Bordetella pertussis (B. pertussis) were assessed by using Pulsed Field Gel Electrophoresis (PFGE). Methods Following phenotypic and molecular identification of isolates, XbaI-digested genomic DNA of 5 clinical isolates, 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Results Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 clinical isolates, 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain, Tohama I had a distinct profile. Vaccine and clinical profiles had low similarity, with relatedness of approximately 40%. Conclusion The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically, the profiles of isolates circulating in the population should be monitored over the course of the re-emergence.