Pertussis is a vaccine preventable disease, primarily caused by the bacterium Bordetella pertussis (and occasionally B. holmesii and B. parapertussis). The disease is highly contagious, and results in extended periods of coughing. Prior to vaccination, pertussis was the foremost cause of infantile death by infection globally. Global vaccination efforts against pertussis were introduced in the 1950s and this resulted in the rate of pertussis infections to decrease tremendously. In recent decades, public health authorities around the world have reported a resurgence of the number of pertussis cases, despite high vaccination coverage. The reason behind this resurgence is unknown, however, there are multiple theories. A major contributing factor is the reduced molecular surveillance of B. pertussis, a consequence of the change in diagnostic methods from culture isolates to direct PCR on clinical samples. Culture isolates enable molecular surveillance by providing information on the strain, such as sequence type and antibiotic resistance information. Without molecular surveillance we know little about the currently circulating strains of B. pertussis. The aims of this thesis were to the provide characterise the genomes of the Bordetella genus. Ultimately, it aimed to attempt explaining the driving forces behind B. pertussis resurgence by identifying their properties of relevance for laboratory diagnosis and surveillance. The thesis also aimed to develop new laboratory protocols to circumvent the lack of isolates and achieve high resolution data and information from clinical samples. It also aimed to determine the differences in the expression of B. pertussis vaccine antigens using metatranscriptomics. Furthermore, the thesis aimed to determine the potential for antimicrobial resistance within the Bordetella spp., and their mechanisms behind resistance. This thesis confirmed through the generation of high-quality closed reference sequences, that Bordetella spp. isolates from NSW carried the same genomic rearrangement characteristics as global isolates. Thus, demonstrating evidence of genomic evolution of the Bordetella spp. in the Southern hemisphere. These reference sequences can serve as a representative of the prevalent strains in the Australian community and be utilised in future public health surveillance. This thesis also describes the development of a protocol to circumvent culture isolates, by combining Saponin depletion with an improved library preparation method, Nextera Flex kit (Illumina) and sequencing so that more reads per sample are generated (i.e. deep sequencing). It demonstrated that with high bacterial load samples, this method can effectively produce the entire genome of B. pertussis with an average 20 X coverage. Therefore, through the utilisation of pre-extraction depletion methods, in conjunction with the proper library preparation kit, and increased sequencing depths, sufficient information can be obtained for molecular surveillance. Culture-independent RNA sequencing was also determined as a feasible method in determining high-resolution strain typing information for public health investigations. Metatranscriptomics was able to show that assemblies could reliably identify segments of the genome coding the vaccine antigens, primarily fhaB and prn. In addition, the thesis identified 10 new genomic markers for further investigation, especially as potential targets for CI-WGS enrichment. The study showed that two of the three Bordetella spp. developed resistance (>256 μg /mL) within 15 weeks upon consistent macrolide exposure. WGS was able to confirm the resistance mutation in B. holmesii – a non-synonymous SNP mutation in the 23S ribosomal RNA (C2585T, G2031A, and A2032G). Thus, the study proved that B. parapertussis and B. holmesii carried a higher capability in developing macrolide resistance under antibiotic pressure in vitro. The mechanism of resistance in these two species was identified in B. holmesii, however remains unclear in B. parapertussis. Overall, the findings of this thesis will facilitate the genomic surveillance of Bordetella spp. in the era of culture-independent diagnostic testing.