Abstract Background Pertussis, an acute respiratory infection caused by Bordetella pertussis ( B. pertussis ), has resurged in China. Biofilm formation has recently been associated with the pathogenesis of B. pertussis . This study aims to analyze the biofilm formation capacity of circulating isolates and investigate the phenotype–genotype associations between biofilm formation and genomic characteristics. Results Among 84 B. pertussis isolates, 69.05% (58/84) were classified as strong biofilm formers, while 23.81% (20/84) and 5.95% (5/84) exhibited moderate or weak biofilm production, respectively. Only one isolate (1/84, 1.19%) was categorized as non-biofilm producers. Strong biofilm-forming isolates exhibited significantly faster autoagglutination than non-strong isolates ( P < 0.05). Pan-GWAS identified multiple candidate genes significantly associated with biofilm phenotypes ( P < 0.05), which were enriched in pathways involved in cell envelope remodeling, metabolic adaptation, nutrient acquisition, transport systems, and environmental sensing. SNP-GWAS further identified multiple core genome SNPs significantly associated with enhanced biofilm formation ( P < 0.05). Functional analysis revealed significant enrichment of genes in functions related to lipoprotein maturation, autotransporter function, amino acid metabolism, and transport systems. Integration of pan-GWAS and SNP-GWAS further identified the genetic determinants correlated with biofilm phenotypic heterogeneity in B. pertussis . Conclusions Strong biofilm-forming isolates consistently exhibited higher levels of biofilm biomass and faster aggregation kinetics than non-strong isolates. GWAS analysis revealed that the observed phenotypic divergence is driven by coordinated remodeling of multiple biological pathways rather than a single genetic determinant. These findings provide new insights into the phenotype–genotype association signatures of biofilm formation in circulating B. pertussis .