AbstractMultidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.