Simple Summary High-quality genome assemblies are essential tools for modern biological research. In the past, creating genome assemblies was prohibitively expensive and time-consuming for most non-model insect species due to, in part, the technical challenge of isolating the necessary quantity and quality of DNA from many species. Sequencing methods have now improved such that many insect genomes can be sequenced and assembled at scale. We created the Ag100Pest Initiative to propel agricultural research forward by assembling reference-quality genomes of important arthropod pest species. Here, we describe the Ag100Pest Initiative’s processes and experimental procedures. We show that the Ag100Pest Initiative will greatly expand the diversity of publicly available arthropod genome assemblies. We also demonstrate the high quality of preliminary contig assemblies. We share arthropod-specific technical details and insights that we have gained during the project. The methods and preliminary results presented herein should help other researchers attain similarly high-quality assemblies, effectively changing the landscape of insect genomics. Abstract The phylum Arthropoda includes species crucial for ecosystem stability, soil health, crop production, and others that present obstacles to crop and animal agriculture. The United States Department of Agriculture’s Agricultural Research Service initiated the Ag100Pest Initiative to generate reference genome assemblies of arthropods that are (or may become) pests to agricultural production and global food security. We describe the project goals, process, status, and future. The first three years of the project were focused on species selection, specimen collection, and the construction of lab and bioinformatics pipelines for the efficient production of assemblies at scale. Contig-level assemblies of 47 species are presented, all of which were generated from single specimens. Lessons learned and optimizations leading to the current pipeline are discussed. The project name implies a target of 100 species, but the efficiencies gained during the project have supported an expansion of the original goal and a total of 158 species are currently in the pipeline. We anticipate that the processes described in the paper will help other arthropod research groups or other consortia considering genome assembly at scale.