Poly (3-hydroxybutyrate) (PHB) is a good alternative to petroleum-based plastics due to its biodegradable and biocompatible properties. In the present work, Cupriavidus necator ATCC 25207 was used for the first time to produce PHB using fructose, glucose, and acid or enzyme hydrolyzed molasses, a cheap carbon source. The bacterium produced highest PHB with 4.99 g/L, 3.94 g/L, and 15.28 g/L using fructose, glucose, and acid hydrolyzed molasses, respectively. Sulfuric acid and invertase treated molasses yielded 1288% and 529% more PHB than untreated molasses. In addition, acid treated molasses resulted in 151% more PHB but 34.04% less cell growth than enzyme hydrolyzed molasses. This novel finding could have applications in fermentations using molasses in growth or production media. The utilization of glucose by the bacterium was increased by 67.03% through spontaneous mutagenesis. The characterizations of the produced polymers were carried out using FTIR, NMR, XRD, DSC, TGA, and GPC. The molecular weights of PHB samples from acid hydrolyzed molasses and fructose are 3.84 x 10(5) and 1.43 x 10(6) g/mol, respectively. This study shows that C. necator ATCC 25207 has a potential to be used in PHB production using acid hydrolyzed molasses.