Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and difficult to treat due to acquired-resistance to multiple antibiotics. A pair of strains, M38100A and M38100B, previously identified from a single clinical isolate of P. aeruginosa was investigated to understand phenotypic and genotypic characteristics. Results revealed that the pair of strains was very similar for serum susceptibility, growth rate in a complex medium (Luria-Bertani), RAPD-genotype profiles, status of genes encoding type III secretion toxins, and no extra-chromosomal DNA. However, antibiotic susceptibility of the strain M38100B showed resistant to all tested-antibiotics while the strain M38100A showed susceptible to the same tested-antibiotics as similar levels of P. aeruginosa PAO1. The strain M38100B exhibited no growth in a minimal medium as a sole carbon and nitrogen source of glutamate while the strain M38100A grew well in the same minimal medium. These results suggest that multidrug resistance of the strain M38100B may be caused by multiple mutations on its genomic DNA and a precursor stage for a homogeneous multidrug resistant population.