The aim of this research was to evaluate the production and properties of polyhydroxyalcanoates (PHAs) obtained from crude glycerin (CG), a byproduct of the biodiesel industry, by Cupriavidus necator IPT 026. Experiments were carried out in shake flasks to determine the optimum CG ( X 1 ) and nitrogen ( X 2 ) concentrations to maximize biomass accumulation and biopolymer production. The highest PHA and biomass production (2.81 g L −1 and 4.34 g L −1 , respectively) occurred at 15 g L −1 of CG and 10 g L −1 of nitrogen with approximately 65% (w/v) cell accumulation (cell dry weight). Experiment 7 yielded the PHA with the optimum properties (15 g L −1 of CG and 3 g L −1 of nitrogen), which showed a melting temperature of 184.3 °C, crystallinity of 52.23%, thermal degradation occurring between 306.8 °C and 334.1 °C with a peak at 327.4 °C, and its molecular weight was 780 kDa. All experiments PHA production showed FTIR spectra similar to those reported in the literature. The monomeric profile of the produced PHAs showed a composition similar to that of the PHA standard, showing evidence of 3-hydroxybutyrate (3HB) monomer in the analyzed samples. Bacteria can use CG as an inexpensive substrate to produce value-added biodegradable products, such as PHA.