The yeast Kluyveromyces marxianusgrows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiaein industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianusstrains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianusas a versatile synthetic biology platform organism for industrial applications.