Trehalose, a naturally occurring, nonreducing disaccharide composed of two glucose units, has been found in a wide variety of species including bacteria, fungi, plants, insects and some invertebrate animals. As a sugar, it has been proposed to serve as a carbon and energy source. In yeast and plants, it also acts as a signaling molecule in regulating certain metabolic pathways. In addition, it has been suggested to protect proteins and cellular membranes against damage or denaturation induced by a variety of stress conditions, such as desiccation, dehydration, heat, cold and oxidation. Its unique physical and chemical properties have also turned it into an attractive ingredient in food, health, cosmetics and pharmaceutical products. However, recent evidence suggests that the protective effects of trehalose on living organisms is condition-specific: accumulating this disaccharide in Saccharomyces cerevisiae cells does not provide heat tolerance whereas intracellular trehalose accumulation does promotes extreme desiccation survival. To further probe the stress conditions in which trehalose directly protects organisms, I examined whether intracellular trehalose accumulation directly provides freeze-thaw tolerance in baker’s yeast, as cryopreservation of intact yeast cells is critical for long-term storage in the baking industry. A genetic system conferring AGT1 overexpression allowed cells to transport and accumulate trehalose inside cell, which is sufficient to provide freeze-thaw tolerance. This can be further enhanced by deleting genes encoding intracellular trehalose degradation enzymes. These findings are relevant to improving the freeze-thaw tolerance of baker’s yeast in the frozen baked goods industry through engineering strains that can accumulate intracellular trehalose via a constitutively expressed trehalose transporter and inclusion of trehalose into the growth medium. Mutants of trehalose metabolism in S. cerevisiae have been extensively characterized in multiple laboratory strains and a panel of unexplained pleotropic phenotypes have been observed. In this work, I constructed mutants in the trehalose metabolic pathway genes (TPS1, TPS2, TPS3, TSL1, NTH1, NTH2, and ATH1) from five different S. cerevisiae genetic background to rigorously investigate whether published laboratory strain phenotypes are also exhibited by wild strains. For each mutant, I assessed trehalose production, glycogen production, cell size, acute thermotolerance, high temperature growth, sporulation efficiency and growth on a variety of carbon sources in rich and minimal medium. Altogether, I comprehensively evaluated and confirmed many of the published phenotypes, expanding this knowledge to include phenotypic variants in multiple strains that could be used to genetically dissect the basis of these trait and then develop mechanistic models connecting trehalose metabolism to various cellular processes. I further identified novel phenotypes associated with tps1∆, nth1∆, nth2∆ and ath1∆ which will contribute to a more comprehensive model for the roles associated with trehalose metabolism.