As a group of winged insects (Pterygota) retaining many primitive characteristics, genomic research on mayflies remains highly limited, posing challenges to the study of their origin and evolution. In this study, we present the first chromosome-level genome assembly of the Chinese endemic mayfly Parafronurus youi utilizing Illumina short-read, PacBio long-read, and Hi-C sequencing technologies. The high-quality genome is 412.90 Mb in size with 99.07% of the sequences anchored to 11 chromosomes (ranging from 24.88 to 45.89 Mb). Genome annotation predicted 15,647 protein-coding genes with an average length of 9,934.7 bp, of which 85.9% were functionally annotated in the UniProtKB database. Repetitive elements accounted for 32.83% of the genome, including 27.33% transposable elements and 4.07% simple repeats. This study not only enriches genomic resources for mayflies but also establishes a foundation for investigating molecular mechanisms underlying ecological adaptation and evolutionary traits, contributing to the conservation of freshwater ecosystems.