IntroductionPseudomonas is one of the largest genera among Gram-negative bacteria, currently comprising nearly 400 valid species. Among them, non-aeruginosa Pseudomonas species, together with Stutzerimonas—a genus recently separated from Pseudomonas—are becoming important reservoirs of antimicrobial resistance genes. However, accurate species-level identification of these organisms remains challenging when relying on the methods routinely implemented in most clinical microbiology laboratories, such as MALDI-TOF mass spectrometry. Therefore, this study aimed to identify and characterize metallo-β-lactamase-producing non-aeruginosa Pseudomonas spp. and Stutzerimonas spp. isolates obtained in a Spanish tertiary hospital, focusing on their species-level identification, dissemination and role as reservoirs of carbapenemase-encoding genes.MethodsWhole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST) were used to identify and characterize a total of 22 carbapenemase-producing non-aeruginosa Pseudomonas and Stutzerimonas isolates collected from patient and environmental samples from March 2022 to July 2024 in a Spanish tertiary hospital.ResultsWGS analysis identified eight species: Pseudomonas alloputida (n = 7), Pseudomonas mosselii (n = 5), Pseudomonas kurunegalensis (n = 4), Pseudomonas asiatica (n = 2), Pseudomonas oleovorans (n = 1), Pseudomonas soli (n = 1), Stutzerimonas chloritidismutans (n = 1) and Stutzerimonas stutzeri (n = 1). Multilocus sequence typing (MLST) classified isolates of the P. putida group into nine STs, revealing three predominant clones: P. alloputida ST69, P. mosselii ST115 and P. kurunegalensis ST114. AST results showed resistance rates >55% to all tested β-lactams, aminoglycosides and fluoroquinolones, except for cefiderocol, amikacin and colistin (0–5%). The following carbapenemase combinations were detected: blaVIM−1 (n = 16), blaVIM−1+blaVIM−2 (n = 4), blaVIM−1+blaNDM−1 (n = 1) and blaVIM−11 (n = 1). Seven distinct blaVIM-harboring plasmids were detected in 16 isolates, whereas the remaining six carried chromosomally-integrated blaVIM. Eight different blaVIM-harboring class I integrons were detected, two of which also occurred in P. aeruginosa clinical isolates obtained in the same hospital.ConclusionThis study highlights the epidemiological relevance of non-aeruginosa Pseudomonas and Stutzerimonas species as reservoirs of metallo-β-lactamase genes encoded in mobile genetic elements. These findings reinforce the need for systematic WGS-based surveillance of both clinical and environmental isolates, enabling the detection of hidden transmission networks and interspecies dissemination of antimicrobial resistance determinants.