The emergence and spread of antimicrobial resistance has become a serious global issue. Bacterial characteristics, such as antimicrobial resistance genes, virulence-associated genes, plasmid types, and phylogenetic relationship among different strains, are the keys to unravel the occurrence and dissemination of antimicrobial resistance. However, the accuracy and efficiency of the traditional techniques, such as polymerase chain reaction and pulsed field gel electrophoresis is insufficient to underlying the mystery of antimicrobial resistance. Recently, the whole genome sequencing and high-throughput bioinformatics analysis have been successfully used in antimicrobial resistance studies, helping scientists to obtain the nature of antimicrobial resistance bacteria quickly, and more precisely to paint the evolutionary relationship among different strains. Therefore, in this study, we aim to systematically introduce the recent development of whole genome sequencing analysis, including different methods and corresponding characteristics of library preparation, platform sequencing, data analysis, and the latest application of the technology in the antimicrobial resistance research. We hope that this review can provide more comprehensive knowledge about whole genome sequencing and bioinformatic analysis for antimicrobial resistance research.