The genus Prototheca, characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii. The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.