BACKGROUND Polyhydroxyalkanoate (PHA) is a biopolymer that can be used as a biodegradable plastic. The incorporation of PHA production into wastewater treatment processes is a promising strategy for resource recovery. This study investigated PHA production in two‐stage continuous stirred tank reactor (CSTR)‐activated sludge (AS) systems fed with synthetic wastewater containing glycerol. The effects of the hydraulic retention time (HRT) ratios of the first and the second stages, which reflects the feast/famine ratio, at 0.04, 0.15, and 0.33 were examined. RESULTS The PHA accumulation and PHA consumption patterns under feast/famine conditions were successfully achieved in systems operated at HRT ratios of 0.04, 0.15, and 0.33 with influent glycerol concentrations of 3000 mg COD/L. Then, the sludge from these systems was tested for PHA accumulation in fed‐batch reactors. Maximum PHA contents of 21.8 wt%, 13.1 wt%, and 8.7 wt% were obtained for the sludge from the systems operated at HRT ratios of 0.04, 0.15, and 0.33, respectively. From the microbial community analysis via 16S rRNA gene amplicon sequencing (MiSeq, Illumina), the relative abundance of betaproteobacteria decreased, whereas the relative abundances of alphaproteobacteria and gammaproteobacteria increased after cultivation in the two‐stage CSTR AS systems. CONCLUSION The highest PHA accumulation was achieved at the lowest HRT ratio of the first and the second stages in the two‐stage CSTR AS systems. This study serves as an example of integrating PHA production into wastewater treatment systems, which enables us to apply the concept of resource recovery from wastewater. © 2019 Society of Chemical Industry