ObjectivesPseudomonas aeruginosahas intrinsic antibiotic resistance and the strong ability to acquire additional resistance genes. However, a limited number of investigations provide detailed modular structure dissection and evolutionary analysis of accessory genetic elements (AGEs) and associated resistance genes (ARGs) inP. aeruginosaisolates. The objective of this study is to reveal the prevalence and transmission characteristics of ARGs by epidemiological investigation and bioinformatics analysis of AGEs ofP. aeruginosaisolates taken from a Chinese hospital.MethodsDraft-genome sequencing was conducted forP. aeruginosaclinical isolates (n = 48) collected from a single Chinese hospital between 2019 and 2021. The clones ofP. aeruginosaisolates, type 3 secretion system (T3SS)-related virulotypes, and the resistance spectrum were identified using multilocus sequence typing (MLST), polymerase chain reaction (PCR), and antimicrobial susceptibility tests. In addition, 17 of the 48 isolates were fully sequenced. An extensive modular structure dissection and genetic comparison was applied to AGEs of the 17 sequencedP. aeruginosaisolates.ResultsFrom the draft-genome sequencing, 13 STs were identified, showing high genetic diversity. BLAST search and PCR detection of T3SS genes (exoT,exoY,exoS, andexoU) revealed that theexoS+/exoU- virulotype dominated. At least 69 kinds of acquired ARGs, involved in resistance to 10 different categories of antimicrobials, were identified in the 48P. aeruginosaisolates. Detailed genetic dissection and sequence comparisons were applied to 25 AGEs from the 17 isolates, together with five additional prototype AGEs from GenBank. These 30 AGEs were classified into five groups -- integrative and conjugative elements (ICEs), unit transposons, IncpPBL16plasmids, Incp60512−IMPplasmids, and IncpPA7790plasmids.ConclusionThis study provides a broad-scale and deeper genomics understanding ofP. aeruginosaisolates taken from a single Chinese hospital. The isolates collected are characterized by high genetic diversity, high virulence, and multiple drug resistance. The AGEs inP. aeruginosachromosomes and plasmids, as important genetic platforms for the spread of ARGs, contribute to enhancing the adaptability ofP. aeruginosain hospital settings.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12941-023-00600-3.