This study aimed to characterise Bordetella pertussis isolates circulating in Poland since 1959. Sequence analysis of ptxA, ptxC, prn, tcfA, fim2, fim3 and ptxP for 175 clinical isolates and currently and previously used vaccine strains was performed. Clinical isolates from the period 1995-2013 were found to be different to three currently used vaccine strains harbouring the allelic combination ptxA2-ptxC1-ptxP1-prn1-tcfA2-fim2-1-fim3-1, seen frequently in Poland in the early pertussis vaccination period but not found after 1995. Generally, among B. pertussis isolates from the period 2000-2013, two genotypes predominated, ptxA1-ptxC1-ptxP1-prn1-tcfA2-fim2-2-fim3-1 and ptxA1-ptxC1-ptxP1-prn2-tcfA2-fim2-1-fim3-1, with frequencies of 45% and 32.5%, respectively. The isolates harbouring ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-2 and ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 profiles, currently highly prevalent within other European Union (EU) countries, were rarely found in Poland, as they circulated in the period 2000-2013 with frequencies of 10% and 5%, respectively. We hypothesise that several previous changes of strain composition in whole-cell pertussis vaccine produced locally and used since 1960 in Poland resulted in a more diverse immune pressure in the population, resulting in different prevalence of alleles compared to elsewhere.