Abstract Background There was a registered increase in the number of confirmed pertussis cases in Central Serbia between June and December 2023. Among the possible causes of pertussis resurgence are increased awareness of pertussis among healthcare workers, availability of the sensitive RT PCR method in diagnostic, the limited duration of vaccine-induced immunity as well as decline of immunization coverage influenced by the COVID-19 pandemic. Methods The nasopharyngeal samples from clinics and primary care departments were transferred to Institute of Public Health of Serbia. DNA was extracted from each sample using the MagMAX Viral/Pathogen kit. The presence of B. pertussis was determined using two PCR assays, VIASURE Bordetella Real Time PCR Detection Kit and Bosphore B. pertussis/parapertussis Genotyping Kit, Anatolia Geneworks. Results RT-PCR confirmed a total of 338 pertussis cases in Institute of Public Health of Serbia during the observed period. Most of confirmed cases were among children 9-14 years old (49,7%) followed by infants younger than 12 months (21,9%). The more severe cases were among the unvaccinated and uncomplete vaccinated infants, 78,4% of which required hospitalization with two deaths in this age group. Conclusions Based on the analysis of the obtained results, we can highlight the importance of using sensitive methods in pathogen detection. Rapid diagnosis, laboratory confirmation and notification ensure early public health intervention to minimize the spread of disease. Despite long tradition of mandatory immunization against pertussis in the Republic of Serbia, wanning of vaccine induced immunity poses potential risk for the resurgence of pertussis. For pertussis control, in the future, it will be essential to achieve high immunization coverage as well as to introduce a booster dose of pertussis containing vaccine for children aged 14 and to implement vaccination of pregnant women. Key messages • For the better pertussis control vaccination of adolescents and adults, as well as vaccination of pregnant women should be considered in the future vaccination policies. • Clinicians should consider pertussis in the differential diagnosis of persistent cough illness in people of all ages, even those previously immunized.