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Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure–function relationships.