Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety ofacute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogencomes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to formbiofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variabil-ity, antimicrobial susceptibility as well as the production of metallo- -lactamases (MBLs) and virulenceattributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatom-ical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, inwhich 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibilityprofile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibi-otics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem onlyand 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistantto both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12(35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastaseand biofilm, although in different levels. Some associations were demonstrated among the susceptibilityand/or production of these virulence traits with the anatomical site of strain isolation. For instance, almostall strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority ofstrains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urineisolates produced the highest pyocyanin concentration (20.15 ± 5.65 g/ml), while strains isolated frompleural secretion and mouth produced elevated elastase activity (1441.43 ± 303.08 FAU) and biofilm for-mation (OD5900.676 ± 0.32), respectively. Also, MBL-positive strains produced robust biofilm comparedto MBL-negative strains. Collectively, the production of site-dependent virulence factors can be high-lighted as potential therapeutic targets for the treatment of infections caused by heterogeneous andresistant strains of P. aeruginosa.