High substrate costs decrease the profitability of polyhydroxyalkanoates (PHAs) production, and thus low-cost carbon substrates coming from agricultural and industrial residuals are tested for the production of these biopolymers. Among them crude glycerol, formed as a byproduct during biodiesel production, seems to be the most promising source of carbon. The object of this study was to characterize the mixed population responsible for the conversion of crude glycerol into PHAs by the cultivation-dependent and -independent methods. Enrichment of the microbial community was monitored by applying the Ribosomal Intergenic Spacer Analysis (RISA) and the identification of community members was based on 16S rRNA gene sequencing of cultivable species. Molecular analysis revealed that mixed populations consist of microorganisms affiliated with four bacterial lineages: alpha, gamma- Proteobacteria, Actinobacteria and Bacteroides. Among them, three Pseudomonas strains and Rhodobacter sp. possessed genes coding for polyhydroxyalkanoates synthase. Comparative analysis revealed that most of the microorganisms detected by direct molecular analysis were obtained by the traditional culturing method.