Biodiesel is a renewable alternative fuel and glycerol as a main byproduct of the manufacturing process. Lipids could be produced from crude glycerol by using yeasts. The ability of 107 yeast strains to utilize glycerol was screened and 92 of these were selected. 60 strains were determined as a potential for lipid production by Sudan Black B staining. After secondary screening 25 of them showed specific growth rates (OD 600), high biomass production and lipid content. These strains were identified as Pichia cactophila, P. fermentans, P. anomala, Rhodotorula mucilaginosa, R. dairenensis, Clavispora lusitaniae, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata, C. inconspicua, C. albicans, Yarrowia lipolytica with molecular identifications based on ITS and D1/D2 26S rDNA sequences. The results showed that P. cactophila accumulated lipid up to 64.94%, the highest lipid content. C16:0, C18:0, C18:1 and C18:2 essential fatty acids for biodiesel production were detected by GC-MS in the lipids accumulated by all strains. P. cactophila and C. lusitaniae were reported for the first time as lipid-producing yeasts. The results suggest that selected 25 isolates have the ability to grow on crude glycerol and especially P. cactophila produce lipid that has potential use as a feedstock for second generation biodiesel production.