ABSTRACTPseudomonas aeruginosaST308 clone has been reported to carry carbapenemase genes such asblaIMPandblaVIMbut has been rarely associated withblaNDM-1. A total of 199P. aeruginosaST308 clinical and environmental isolates obtained between April 2019 and November 2020 from a tertiary-care hospital in Singapore were characterized using whole-genome sequencing. In addition, 71blaNDM-1-positive ST308 whole-genome sequences from two other local tertiary-care hospitals in Singapore and 83 globalblaNDM-1-negative ST308 whole-genome sequences in public databases were included to assess phylogenetic relationships and perform genome analyses. Phylogenetic analysis and divergent time estimation revealed thatblaNDM-1-positiveP. aeruginosaST308 was introduced into Singapore in 2005 (95 % highest posterior density: 2001 to 2008). Core genome, resistome, and analyses of all localblaNDM-1-positive ST308 isolates showed chromosomal integration of multiple antibiotic resistance genes (ARGs) [aac(3)-Id,aac(6′)-Il,aadA6,aadA11,dfrB5,msr(E),floR,sul2, andqnrVC1], which was absent in globalblaNDM-1-negative ST308 sequences. Most ARGs and virulence genes were conserved across isolates originating from the three different local hospitals. Close genetic relatedness of theblaNDM-1-positive ST308 clinical and environmental isolates suggests cocirculation between the hospital environment and human hosts with the hospital environment as a potential reservoir. Core genome single nucleotide polymorphism analyses revealed possible clonal transmission ofblaNDM-1-positive ST308 isolates between the three hospitals over 7 years. Bloodstream isolates accounted for six of 95 (6.3%) clinical isolates. This study reports the introduction of a pathogenicblaNDM-1-positiveP. aeruginosaST308 more than a decade ago in Singapore and warrants surveillance for wider dissemination.IMPORTANCEP. aeruginosais a Gram-negative opportunistic pathogen ubiquitously found in the environment and a major cause of nosocomial infections. While theP. aeruginosaST308 clone has been known to bearblaIMPandblaVIMamong global isolates, reports ofblaNDM-1-positiveP. aeruginosaST308 are rare. The localblaNDM-1-positiveP. aeruginosaST308 isolates detected in this study appear to be unique to this region, with evidence of chromosomal acquisition of multiple ARGs compared to globalblaNDM-1-negativeP. aeruginosaST308 isolates. Surveillance in Singapore and beyond for dissemination is essential to determine whether existing measures are sufficient to control the spread of this ST308 clone.