In spite of aggressive treatment with antibiotics and the immune host defense, once established Pseudomonas aeruginosa persists in the Cystic Fibrosis (CF) airways for years or even decades. One cause of its persistence is the capacity to grow as biofilms that provide increased survival ability under a variety of stress conditions. Contributing to this, high prevalence of hypermutable Mismatch Repair System deficient isolates have been observed in CF chronic lung infection. It has been proposed that hypermutators play an important role in the acquisition of adaptative mutations needed for long-term survival of this bacterium in the CF airways. Despite the potential importance of an increased mutagenesis in the persistence of P. aeruginosa CF infections, the effect of hypermutation in biofilm formation has been poorly investigated. In this sense, the aim of the present study was to investigate the effects of hypermutator phenotype on the architecture and phenotypic diversification of P. aeruginosa biofilms. For this purpose, the reference strain PAO1, environmental strain Hex1T and their respective mutS mutants, PAOMS and Hex1TMS, were used. Biofilms were grown at 30°C in three-channel flow cells and pictures were taken at 2, 4 and 6 days. To study the phenotypic diversification, 6 days-biofilms were harvested, plated on P. aeruginosa isolation agar and the morphologies of approx. 2.000 colonies per strain were examined. All microscopic observations were performed by a confocal laser scanning microscope (CLSM). For structural analysis and processing of images software packages COMSTAT and IMARIS were used. Structural data were compared using a Student t test. In this work, we show that biofilms formed by PAOMS were thicker and showed more biomass than those formed by PAO1 (P < 0.05 at 4d). However, although no significant differences were found at days 2 and 4, Hex1TMS biofilms were thinner and showed less biomass than Hex1T at 6d (P < 0.05). CLSM images of biofilms formed by hypermutable strains showed irregular biofilms structures, randomly smooth, rough and filamentous. Empty mushroom-like structures were characteristically observed in Hex1TMS biofilms. Examination of colony morphology after 6d-biofilm revealed up to 6 different morphotypes in hypermutable and only 2 within wild-type strains. Normal, giant, mini, wrinkly, mucoid, brown and orange pigmented were among the different morphotypes found. Total percentages of colonies with different morphotypes were 8.3 and 0.05 % for PAOMS and PAO1, respectively, and 2.3 and 0.8% for Hex1TMS and Hex1T, respectively. The results presented here demonstrated that Mismatch Repair System deficiency increases the structural diversity of P. aeruginosa biofilms and this could probably be a microscopically visible evidence of the acceleration of phenotypic diversification.