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Infections with multidrug-resistant bacteria often leave limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we demonstrate the application of Nanopore sequencing in a hospital setting for monitoring transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species. In 2009, we experienced an outbreak with extensively multidrug-resistant Pseudomonas aeruginosa harboring the carbapenemase-encoding blaIMP-8 gene. In 2012, the first Citrobacter freundii and Citrobacter cronae strains harboring the same gene were detected. Using Nanopore and Illumina sequencing, we conducted comparative analysis of all blaIMP-8 bacteria isolated in our hospital over a 6-year period (n = 54). We developed the computational platform plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de novo assembly of genomes and plasmids, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates, and visualization of results. Using plasmIDent, we identified a 40-kb plasmid carrying blaIMP-8 in P. aeruginosa and C. freundii, verifying the plasmid transfer. Within C. freundii, the plasmid underwent further evolution and plasmid fusion, resulting in a 164-kb megaplasmid, which was transferred to C. cronae. Multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs. In summary, plasmid transfer, plasmid fusion, and rearrangement of the ARG cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of near-real-time monitoring of plasmid evolution and ARG transfer in clinical settings, enabling successful countermeasures to contain plasmid-mediated outbreaks. IMPORTANCE Infection, We thank Nadine Hoffmann and Baris Bader for expert technical assistance. The study was conducted in accordance with the local ethic committee (Ethic Committee Medical Faculty, University of Tübingen, No. 741/2016BO2 and 407/2013R). We declare that we have no competing interests. S.P. received a speaker’s honorarium from bioMérieux, Deutschland, and consultancy honoraria from IDbyDNA, San Francisco, CA, USA, and Illumina, Cambridge, United Kingdom, which had no role in the design, analysis, interpretation, and writing of the manuscript or in the decision to publish the data. The study was funded by the Faculty of Medicine of the University of Tübingen, the Spanish Ministry of Economy and Competitiveness, the Centro de Excelencia Severo Ochoa, the CERCA Program/Generalitat de Catalunya and the “la Caixa” Foundation. The funders had no role in the design, analysis, interpretation, and writing of the manuscript. Software development and bioinformatics analysis were performed by M.B. during the first half of the 3-year project and by C.G. during the second half. S.P., D.B., P.O., J.G., M.M., M.W., I.G., and M.G. generated the laboratory and sequencing data. J.L., W.V., and D.D. gathered epidemiological data. S.P., M.B., C.G., and S.O. performed the data analysis. C.G., M.B., and S.O. developed the bioinformatics methods and pipelines, and L.B. developed the GU, Peer Reviewed, Postprint (published version)