To evaluate the presence of biofilm-specific antibiotic-resistant genes, PA0756-0757, PA5033 and PA2070 in Pseudomonas aeruginosa isolated from clinical samples in Tamil Nadu. For this cross-sectional study, 24 clinical isolates (included pus, urine, wound, and blood) were collected from two diagnostic centres in Chennai from May 2015 to February 2016. Biofilm formation was assessed using microtiter dish biofilm formation assay and minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) were determined for planktonic and biofilm cells (MBC assay). Further, PCR amplification of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033 and PA2070 were performed. Biofilm formation was found to be moderate/strong in 16 strains. MBC for planktonic cells showed that 4, 7, 10 and 14 strains were susceptible to gentamicin, ciprofloxacin, meropenem and colistin respectively. In MBC assay for biofilm cells (MBC-B), all the 16 biofilm producing strains were resistant to ciprofloxacin and gentamicin whereas nine and four were resistant to meropenem, and colistin respectively. The biofilm-specific antibiotic-resistant genes PA0756-0757 was found in 10 strains, 6 strains with PA5033 and 9 strains with PA2070 that were found to be resistant phenotypically. This study highlighted the importance of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033, and PA2070 in biofilm-forming P. aeruginosa.