Abstract Polymers are materials of pronounced importance in the modern world, since they are massively present in everyday life, especially in the form of packaging. However, most of these materials are derived from non-renewable sources and their disposal generates large volumes of waste, which is extremely damaging to natural environments. In this context, microbial biopolymers appear as a powerful alternative in the substitution of several applications of synthetic plastics, causing less harm to the environment, as they are biodegradable and produced from renewable sources. In this study, evaluation and characterization of polyhydroxyalkanoates (PHAs) produced by Cupriavidus necator (IPT 026 and IPT 027) and Burkholderia cepacia (IPT 119 and IPT 400), using crude glycerol as substrate, were carried out (crude glycerol 15 g L-1, pH 7.0, 150 rpm, 72h). The substrate chemical composition was determined and all microorganisms tested were able to utilize it to synthesize PHA. C. necator IPT 026 exhibited the highest polymer production (1.52 ± 0.03 g L-1). B. cepacia strains produced low crystallinity PHA. All polyesters synthesized exhibited long chain length polymers with low polydispersity and initial thermal degradation temperatures superior to 300°C. The microorganism strains and the substrate composition highly affected PHAs synthesis, composition and thermochemical characteristics.