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Background: Trimethoprim/sulfamethoxazole (TMP/SMZ), traditionally a second-line drug, is now proposed as the primary alternative treatment for macrolide-resistant Bordetella pertussis (B.pertussis). Thus, there is an urgent need to evaluate the phenotypic profile of B. pertussis regarding susceptibility to TMP/SMZ., Methods: Kirby-Bauer (KB) disk diffusion methods and E-test strips were employed to assess the in vitro susceptibility profiles of 128 B. pertussis strains isolated in China between 2017 and 2022 against TMP/SMZ. Additionally, whole genome sequencing was performed on one strain exhibiting a heterogeneous phenotype., Results: B. pertussis displayed three phenotypes to TMP/SMZ based on the first 10 days of culture: a predominant fuzzy-bordered phenotype (118/128, 92.2 %), a minority with a clear-bordered phenotype (8/128, 6.3 %), and a sporadic heterogeneous phenotype (2/128, 1.6 %) where TMP/SMZ heterozygous colonies emerged on the 9th day of incubation. The diameter of the inhibition zone around the paper disk will decrease as the culture time extends, while the MIC value will change in the opposite direction. Almost in all tests, the bacterial colony will cover the entire inhibitory zone after 10-14 days of incubation. Strains exhibiting heterogeneity were compared with the complete genomes of Tohama I and CS strains, and 191 and 179 point mutation sites were identified, respectively, with multiple variants associated with the expression of transcriptional regulators., Conclusion: This study highlights the diverse drug sensitivity phenotypes of B. pertussis to TMP/SMZ and the importance of uniform testing protocols. Continuous and dynamic monitoring of B. pertussis isolates for TMP/SMZ sensitivity, along with investigating its correlation with epidemiological changes, is essential for establishing a scientific foundation for the effective management and control of pertussis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)