Thermotolerant bacteria producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) were isolated from various thermal sites, including palm oil mill effluent, textile wastewater, and hot spring water, in Thailand. Fifteen strains were isolated at 45 degrees C using nutrient-rich (NR) medium. However, only six isolates produced mcl-PHA at 0.41 +/- 0.01 g/L to 0.80 +/- 0.01 g/L, representing a mcl-PHA content of 29.44% to 50.77% of the dry cell weight (DCW). The six strains of bacterial isolates could utilise a variety of substrates; all were identified asBacillus thermoamylovorans. The highest mcl-PHA content (50.77% of the DCW) was accumulated by theB. thermoamylovoransstrain PHA005 isolated from palm oil mill effluent. The mcl-PHA from strain PHA005 was composed of five different monomers, 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxytetradecanoate (3HTD), 3-hydroxyhexadecanoic acid (3HHD), and 3-hydroxyoctadecanoic (3HOD), with a monomer content of 24.12, 15.50, 13.00, 39.25, and 8.13 mol%, respectively. The optimum temperature forB. thermoamylovoransstrain PHA005 growth is 45 degrees C, and it can survive at up to 60 degrees C. This is a first report of PHA synthesis by a thermotolerantB. thermoamylovorans. Moreover, the high content of 3HHD monomers (39.25 mol%) has never been reported inBacillus.