Background: Pseudomonas aeruginosa, is the most common pathogen in patients with cystic fibrosis (CF) that shows various resistance to antibiotics, acquires mucoidity and multiple genotypes. This survey was performed to study phenotypic and genotypic variations among P. aeruginosa isolates in CF patients at Alzahra Hospital in Isfahan, Iran. Materials and Methods: The isolates of Pseudomonas aeruginosa from CF patients at Alzahra Hospital was identified by appropriate biochemical and microscopic tests, then performed antibiotic resistance tests and mucoid colony morphotyping. The genum of isolates extracted and confirmed on 16S rDNA-based PCR assay and typed on 16S rDNA-23SrDNA spacer, restricted with Hinf1 restriction enzyme. Results: P. aeruginosa was isolated from 21 of the 59 CF patients (35.5%), Out of 21 isolates 9 (42.8%) strains were revealed mucoid morphotype. 81.8% isolates of mucoid strains were resistance to at least one of four antibiotics (GM, AN, PIP and CP). Most of the isolates (86%) showed resistance to ceftazidime. Ribotyping revealed two patterns (P1, P5). Conclusion: The isolates of P. aeruginosa showed meaningful difference between drug resistance to antibiotics. The majority of P. aeruginosa isolated from CF patients showed pattern1 of PCR-Ribotyping.