In Pseudomonas aeruginosa, conversion to the mucoid phenotype marks the onset of an irreversible state of the infection in Cystic Fibrosis (CF) patients. The main pathway for mucoid conversion is mutagenesis of the mucA gene, frequently due to −1 bp deletions in a simple sequence repeat (SSR) of 5 Gs (G5-SSR426). We have recently observed that this mucA mutation is particularly accentuated in Mismatch Repair System (MRS)-deficient cells grown in vitro. Interestingly, previous reports have shown a high prevalence of hypermutable MRS-deficient strains occurring naturally in CF chronic lung infections. Here, we used mucA as a forward mutation model to systematically evaluate the role of G5-SSR426 in conversion to mucoidy in a MRS-deficient background, with this being the first analysis combining SSR-dependent localized hypermutability and the acquisition of a particular virulence/persistence trait in P. aeruginosa. In this study, mucA alleles were engineered with different contents of G:C SSRs, and tested for their effect on the mucoid conversion frequency and mucA mutational spectra in a mutS-deficient strain of P. aeruginosa. Importantly, deletion of G5-SSR426 severely reduced the emergence frequency of mucoid variants, with no preferential site of mutagenesis within mucA. Moreover, although mutagenesis in mucA was not totally removed, this was no longer the main pathway for mucoid conversion, suggesting that G5-SSR426 biased mutations towards mucA. Mutagenesis in mucA was restored by the addition of a new SSR (C6-SSR431), and even synergistically increased when G5-SSR426 and C6-SSR431 were present simultaneously, with the mucA mutations being restricted to −1 bp deletions within any of both G:C SSRs. These results confirm a critical role for G5-SSR426 enhancing the mutagenic process of mucA in MRS-deficient cells, and shed light on another mechanism, the SSR- localized hypermutability, contributing to mucoid conversion in P. aeruginosa.