Introduction: <i>Pseudomonas putida</i> is well known as a potential cell factory for many different biochemicals. Biofilm-based production can be advantageous for possibly toxic products due to increased chemical tolerance and robustness. Biofilm cells frequently differentiate, which challenges the benefits of biofilm-based production, and knowledge about factors driving the heterogeneity is therefore of importance.Methods: Biofilm flow chamber systems connected to confocal laser scanning microscopy were used to study biofilm structures of<i> P. putida</i> KT2440 at different carbon conditions. Subsequent plating of mature biofilm allowed for variant selection followed by pheno- and genotypic analysis.Results: Structure and cell differentiation in mature <i>P. putida</i> KT2440 biofilms were highly dependent on the type of carbon source utilised. Low glucose concentrations (0.3 mM – 10 mM) did not alter biofilm structures nor motility, biofilm capability nor growth rate. However, increasing the glucose concentration (15 - 30 mM) introduced filamentous cell structures, as well as variations in colony morphology. Filamentous structures were also observed when using citrate (1 mM – 50 mM), and different morphotypes appeared. These morphotypes showed a large variation in swimming motility, biofilm formation and growth rate, and whole genome sequencing revealed alterations in cyclic-di-GMP-related genes involved in biofilm development.Conclusions: Low concentrations of glucose seem to allow a more homogenous <i>P. putida </i>KT2440 biofilm. In contrast, citrate as a carbon source induces cell differentiation and seems associated with an apparent selection for mutations of genes involved in cyclic-di-GMP-mediated global regulation.