In recent times, the global economy has been facing growing food and energy demands, largely due to fast population growth and an increasingly unpredictable climate. While several multifaceted endeavors are being undertaken to alleviate these demands, this thesis proposes a biorefinery approach that contributes to the collective solution, where methanol is converted to lipids via microbial pathways. Having long been a common industrial feedstock, methanol is becoming even more appealing due to technologies that allow for its sustainable and efficient production. On the other hand, along with those extracted from animals and plants, microorganisms are a great source of lipids that has not been fully exploited. Lipids are versatile precursors for many essential commodities, and creating new pathways for their production can help satisfy the ever-increasing demand for food and energy. As such, this research investigated methods to bring methanol and microbial lipids together in an effort to uplift their industrial potential, as well as to contribute to the scientific understanding of oleaginicity in methylotrophic microorganisms. In the first stage of this project, several known microorganisms were screened to find ideal candidates to perform the methanol-to-lipid conversion. With no suitable hosts identified, the second stage undertook a bioprospecting approach, where microorganisms were isolated from several promising habitats that could potentially harbor oleaginous methylotrophs. A strain collection of 82 isolates was obtained, among which 70 showed signs of intracellular carbon accumulation, as indicated by a lipid-binding dye called Sudan Black B. Almost all isolates that showed Sudan Black B retention were obtained from wetlands or oil sands-related environments, both of which are known to contain a disproportionately higher concentrations of carbon sources, such as methanol, methane, hydrocarbons, carbohydrates, and organic polymers, compared to other nutrients, making them ideal environments for carbon-accumulating methylotrophs. The major genera found were Hyphomicrobium and Ancylobacter, both belonging to the Alphaproteobacteria class. Gas chromatography analysis of selected isolates revealed that isolates Methylorubrum extorquens OSD20, Hyphomicrobium spp. SdF61, OSCF32, and OSFP11 can accumulate approximately 20% or more of their fatty acids as cell dry weight, making them oleaginous methylotrophs. Hyphomicrobium sp. OSFP11 is a potential production host for methanol-to-lipid conversion, thanks to its high proportion of long-chain fatty acids, which were the desirable products in this research (89% of total fatty acids). M. extorquens OSD20 and Hyphomicrobium sp. SdF61, on the other hand, produced significant levels of shortchain fatty acids (30% and 17% of total fatty acids, respectively). As the final stage of this project, the effects of culture conditions on isolates’ fatty acid profiles were examined. Compared to solid culture on agar, Hyphomicrobium sp. OSFP11’s butenoic acid (C4:1) proportion in its fatty acid profile was reduced from 11.1% (on agar) to almost zero (in liquid culture), while its long-chain fatty acid level increase from 67.6% on agar to 82.4% in liquid culture. Hyphomicrobium sp. OSCF32 saw a reverse trend, with more C4:1 and less long-chain fatty acids in liquid culture compared to agar culture. This isolate was also able to produce a substantial level of dihydrosterculic acid, a cyclopropane fatty acid on agar (23% of fatty acid profile), which however, was reduced to 5% in liquid culture. Cold (5 °C) and warm (45 °C) incubation temperatures were found to significantly alter isolates’ fatty acid profiles. Compared to 30.8% of all fatty acids at the 30 °C control condition, M. extorquens OSD20 saw a reduction in its short-chain fatty acid proportion to 22.5% at both 5 °C and 45 °C, and an increase in long-chain fatty acids (from 58.8% at 30 °C to 70.6% at 5 °C, and 67.2% at 45 °C). Overall, this research identified and characterized various potentially novel methylotrophic bacteria that can accumulate significant levels of lipids as well as polyhydroxyalkanoates. At the same time, it revealed further knowledge about methylotrophic populations in understudied environments, thereby contributing to a strong foundation for further research on native methylotrophic microorganisms and their applications.