Next-generation sequencing has been increasingly used to investigate the evolution of pathogenic bacteria, owing to its high throughput and accuracy. NGS can detect internal genetic mutations accumulated in the genomes of bacterial pathogens during their colonization in the hosts, whereas it can also identify the acquired antimicrobial resistance determinants from external sources via horizontal gene transfer (HGT).Both internal genetic mutations and HGT can significantly contribute to the arising of new traits in microbes, and therefore, are important for the adaptive evolution of pathogenic bacteria.In this thesis, we employed both Pacific Biosciences and Illumina sequencing methods to study the evolution of two pathogenic bacteria, Pseudomonas aeruginosa and Salmonella enterica subsp. enterica (S. enterica). We first investigated the in vivo evolution of P. aeruginosa by analyzing the internal genetic mutations in the genomes of longitudinally sampled P. aeruginosa from four ventilator-associated pneumonia (VAP) patients. We conclude that the in vivo evolution of P. aeruginosa shows convergence across the four VAP patients and leads to attenuated virulence of the pathogen. We further identified a novel integrative and conjugative element ICE Tn4371 6385 from a clinically isolated P. aeruginosa strain in Singapore.The acquisition of ICE Tn4371 6385 by the host causes resistance to carbapenem and macrolide-mediated quorum sensing inhibition, both of which are important therapies for P. aeruginosa infections. And finally, we fully characterized a novel IncHI1/N plasmid pSGB23 from a S. enterica strain isolated from a local food market.Strikingly, pSGB23 encodes 12 antimicrobial resistance genes conferring resistance to 10 classes of antimicrobials on its hosts and is self-transmissible among