The resurgence of pertussis also known as whopping cough has been reported worldwide including Australia. Strain variation and pathogen adaptation have been reported in many countries in response to acellular pertussis vaccine. Most recently Single Nucleotide Polymorphism (SNP) typing was used and separated Australian B. pertussis isolates into five clusters, known as SNP cluster I to V with the current predominant cluster I strains, also known as the ptxP3 strains. Whole genome sequencing was used to investigate the microevolution of 22 B. pertussis isolates from the latest Australian pertussis epidemic (2008-2012), which all belonged to SNP profile 13 of cluster I. Ten of the 22 isolates were pertactin (Prn) negative with three different mechanisms of inactivation. Five Australian pre-epidemic isolates, all Prn positive, were also included for analyses. There were five SNPs differentiating epidemic isolates from pre-epidemic isolates. Phylogenetic analysis separated the 22 epidemic isolates into 5 lineages, EL1 to EL5. There were spatial and temporal clustering for the isolates analysed. However, there were also some isolates from different locality and time of isolation that were grouped together suggesting clonal spread of B. pertussis across Australia. Similarly, one of the seven isolate with the same prn gene inactivation were separated from the remaining six suggesting independent evolution of Prn negative strains. The overall genomic diversity and molecular evolution among major Australian clones were then investigated using Illumina and PacBio sequencing. The results confirmed the previous SNP clusters and showed ongoing genome reduction with the deletion of two large regions of differences including BP0910-BP0934 in all clusters and BP1947-BP1968 specific to cluster I. Our findings also revealed the role of progressive gene loss, frameshift indels, new insertion elements and genome arrangements which may have contributed to the evolution and divergence of B. pertussis isolates. Lastly, a mixed infection competition assay in a mouse model study was used to determine the differential fitness between Prn negative and Prn positive strains representing the predominant cluster I as well as between cluster I and cluster II strains. A novel tagged primer Illumina sequencing was used to differentiate the proportion of each isolate in the extracted DNA from lungs and trachea of control and ACV-immunised mice. The results revealed that cluster I strains colonised better in mice respiratory tract regardless of immunisation status and Prn negative strains have better fitness in ACV-immunised mice.