Cold tolerant sugarcane (Saccharum spp.) cultivars are important in sub-tropical production areas because they can extend the growing and harvest seasons, and increase ratooning ability. Improved cold tolerance in Saccharum species has the potential to expand the range of adaptation of sugarcane, thus facilitating its use as a biofuel feedstock in non-traditional cane-growing areas. Selection for this trait is difficult in temperate regions because damaging freezes do not occur every year. The Objective of this study was to use a bioassay to identify sources of cold tolerance among Saccharum spontaneum accessions for use in future breeding. A total of 41 S. spontaneum accessions were evaluated for survivability of below ground (stubble) buds following exposure to freezing temperatures using a growth chamber. Pots containing stubble were frozen for six days at -7 degrees C, and bud germination was determined three weeks post-treatment. Accessions identified as having more ratoon cold tolerance than the most tolerant tested commercial variety (HoCP 96-540) were IND 81-144, IND 81-80, IND 81-165, and MPTH 97-216. These clones will be used in future crossing efforts, and their progeny will be evaluated for cold tolerance under natural conditions in colder climates. Published by Elsevier Ltd.