The cyanobacterium Synechocystis sp. PCC 6803 is known for producing polyhydroxybutyrate (PHB) under unbalanced nutrient conditions. Although many cyanobacteria produce PHB, its physiological relevance remains unknown, since previous studies concluded that PHB is redundant. In this work, we try to better understand the physiological conditions that are important for PHB synthesis. The accumulation of intracellular PHB was higher when the cyanobacterial cells were grown under an alternating day–night rhythm as compared to continuous light. In contrast to previous reports, a reduction of PHB was observed when the cells were grown under conditions of limited gas exchange. Since previous data showed that PHB is not required for the resuscitation from nitrogen starvation, a series of different abiotic stresses were applied to test if PHB is beneficial for its fitness. However, under none of the tested conditions did cells containing PHB show a fitness advantage compared to a PHB-free-mutant (ΔphaEC). Additionally, the distribution of PHB in single cells of a population Synechocystis cells was analyzed via fluorescence-activated cell sorting (FACS). The results showed a considerable degree of phenotypic heterogeneity at the single cell level concerning the content of PHB, which was consistent over several generations. These results improve our understanding about how and why Synechocystis synthesizes PHB and gives suggestions how to further increase its production for a biotechnological process.