Multidrug-resistant Pseudomonas aeruginosa (MDRPA) infections are major threats to healthcare-associated infection control and the intrinsic molecular mechanisms of MDRPA are also unclear. We examined 348 isolates of P. aeruginosa, including 188 MDRPA and 160 non-MDRPA, obtained from five tertiary-care hospitals in Guangzhou, China. Significant correlations were found between gene/enzyme carriage and increased rates of antimicrobial resistance (P < 0·01). gyrA mutation, OprD loss and metallo-β-lactamase (MBL) presence were identified as crucial molecular risk factors for MDRPA acquisition by a combination of univariate logistic regression and a multifactor dimensionality reduction approach. The MDRPA rate was also elevated with the increase in positive numbers of those three determinants (P < 0·001). Thus, gyrA mutation, OprD loss and MBL presence may serve as predictors for early screening of MDRPA infections in clinical settings.