Plasmids are major drivers of increasing antimicrobial resistance (AMR) worldwide. Thus, understanding the biology of plasmids, including mechanisms of replication and maintenance within bacterial cells and transfer to recipient cells, is fundamental for the development of new approaches to combat AMR. Here, we employed in vitrotransposon mutagenesis combined with Transposon-Directed Insertion-site Sequencing (TraDIS) to determine the genetic mechanisms of replication, maintenance and transfer of AMR plasmids from three different groups (IncF, IncC, I-complex). In terms of replication and maintenance, our analyses revealed (i) howcooperation between different replicons on the same plasmid can ensure stable inheritance over multiple generations, (ii) active toxin-antitoxin systems, (iii) functional partition genes, and (iv) novel genes that impact plasmid stability. We further showed how this knowledge can be used to design a plasmid multilocussequence typing (PMLST) scheme based on biologically validated targets to track plasmid dissemination. In terms of conjugation, our approach: (i) accurately identified known genes, (ii) discovered new regulatory and accessory genes that control plasmid transfer, and (iii) identified new genes in the leading transfer region that must be tightly silenced to avoid deleterious effects in the recipient cell. While some of these findings relate to specific plasmids, the interpretations have broad relevance across the diverse classes of AMR plasmids. Overall, our work highlights the powerof TraDIS as an experimentally validated high-resolution screen that can be used to enhance our understanding of AMR plasmid biology, a critical step in the quest to reduce the spread of AMR.