C-di-GMP is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. While the molecular mechanisms of the c-di-GMP signaling have been well studied, the differential distribution of c-di-GMP across biofilms is less well understood. An in-situ, ratiometric, image-based quantification method was developed here to monitor c-di-GMP distribution in flowcell biofilm based on expression of the green fluorescence protein under the control of the c-di-GMP responsive cdrA promoter. Microcolonies smaller than 50 mm in diameter showed a uniform distribution of c-di-GMP. In contrast, for microcolonies that were bigger than 50 mm in diameter, the c-di-GMP appeared to be localized to the boundary of the microcolonies while the interiors showed little fluorescence, suggesting low concentrations of c-di-GMP. The monitor strain was also used to characterize changes in c-di-GMP when the biofilm perceives a dispersal signal, in this case, starvation. Interestingly, after 3 days of continuous starvation induced dispersal, 26% of the biofilm failed to disperse and displayed high reporter fluorescence, suggesting those cell contained high concentrations of c-di-GMP. Isolation and genetic characterization of these cells lead to the conclusion that these colonies that failed to disperse upon carbon starvation were mutants, primarily in wspF, pilT, and fha1 as determined by genome sequencing. Furthermore, LC-MS/MS based quantification indicated that these mutants also had higher intracellular concentrations of c-di-GMP compared to the parent strain. The method was also successfully applied to an experimental mixed species biofilm community and showed that c-di-GMP expression patterns were similar in communities as well as populations. Thus, based on the visualization of c-di-GMP using the ratiometric method described here, it is possible to map relative differences in c-di-GMP heterogeneity in biofilms.