The multifactorial nature ofPseudomonas aeruginosabiofilm development and genomic variabilities implicates its resistance to conventional antimicrobials and virulence. Therefore, genetic determinants need to be extensively studied to block the early steps of biofilm or already formed biofilms. In this study, a total of 20 multidrug resistant (MDR) clinicalP. aeruginosaisolates were evaluated for their biofilm forming abilities and related genes. Of the isolates tested, all of them showed surface attachment tendencies in nutrient limiting conditions, and classified as strong (SBF = 45%), moderate (MBF = 30%) and weak (WBF = 25%) biofilm formers. Complete genome sequencing of representative strong (DMC-27b), moderate (DMC-20c) and weak biofilm former (DMC-30b) isolates was performed. Analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98–100% sequence identity to the reference PAO1 strain. Complete and partial sequence data of LecB proteins from tested isolates indicate that isolates containing PA14-like LecB sequences produced strong biofilms. All of the 7peloperon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide sequence variation with other tested isolates, and their corresponding proteins are 99% identical with thepeloperon proteins of PA7. Bioinformatics analyses identified divergent sequence and structural features that separate PA7 likepeloperon proteins from reference PAO1-likepeloperon. Congo red and pellicle forming assays revealed that the sequence and structure variations may have interfered with the Pel production pathway and resulted in impaired Pel production in isolate 30b that has a PA7 likepeloperon. Expression analysis also showed that bothpelB andlecB genes were about 5 to 6 folds upregulated after 24 h in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes ofP. aeruginosastrains that affect their biofilm phenotypes.