Hemp seeds aqueous extract (HSAE) was used as cheap renewable feedstocks to grow novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 isolated from Himalayan permafrost soil. The yeast showed boosted triglyceride (TAG) accumulation in the lipid droplets (LDs) which were transesterified to biodiesel. The sonicated HSAE prepared lacked toxic inhibitors and showed enhanced total lipid content and lipid yield 55.56%, 8.39 +/- 0.57 g/l in comparison to 41.92%, 6.2 +/- 0.8 g/l from industrially used glucose synthetic medium, respectively. Supersized LDs (5.95 +/- 1.02 lm) accumulated maximum TAG in sonicated HSAE grown cells were visualized by fluorescent BODIPY (505/515 nm) stain. GC-MS analysis revealed unique longer carbon chain FAME profile containing Arachidic acid (C-20:0) 5%, Behenic acid (C-22:0) 9.7%, Heptacosanoic acid (C-27:0) 14.98%, for the first time in this yeast when grown on industrially competent sonicated HSAE, showing more similarity to algal oils. Crown Copyright (C) 2014 Published by Elsevier Ltd. All rights reserved.