1,3-propanediol (1,3-PD) is the important product used in chemical industry. Microbiological synthesis of 1,3-PD from crude glycerol is a good solution, both from an economic and environmental point of view. The aim of this work was to investigate the effect of raw material (pure and crude glycerol) on the efficiency of the synthesis of 1,3-PD by the bacteria Clostridium butyricum DSP1 and Clostridium butyricum DO14 isolated from the samples taken from natural environment. Two strains of C. butyricum were simultaneously investigated. The obtained results showed that the concentration of 1,3-PD was slightly lower in the case of crude glycerol than in pure glycerol, for both strains. Moreover, waste glycerol was not completely utilized.