Aims: To investigate the use of confocal Raman microspectroscopy ( CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. Methods and Results: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium ( artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances ( EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in- plane and - depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology characteristic of the seeding dispersal process - were also observed. Conclusions: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. Significance and Impact of the Study: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.