AbstractPrunus pusilliflorais a wild cherry germplasm resource distributed mainly in Southwest China. Despite its ornamental and economic value, a high-quality assembledP. pusillifloragenome is unavailable, hindering our understanding of its genetic background, population diversity, and evolutionary processes. Here, wede novoassembled a chromosome-scaleP. pusillifloragenome using Oxford Nanopore, Illumina, and chromosome conformation capture sequencing. The assembled genome size was 309.62 Mb, with 76 scaffolds anchored to eight pseudochromosomes. We predicted 33 035 protein-coding genes, functionally annotated 98.27% of them, and identified repetitive sequences covering 49.08% of the genome. We found thatP. pusilliflorais closely related toPrunus serrulataandPrunus yedoensis, having diverged from them ~41.8 million years ago. A comparative genomic analysis revealed thatP. pusilliflorahas 643 expanded and 1128 contracted gene families. Furthermore, we found thatP. pusilliflorais more resistant toColletotrichum viniferum,Phytophthora capsici, andPseudomonas syringaepv.tomato(Pst) DC3000 infections than cultivatedPrunus avium.P. pusillifloraalso has considerably more nucleotide-binding site-type resistance gene analogs thanP. avium, which explains its stronger disease resistance. The cytochrome P450 and WRKY families of 263 and 61 proteins were divided into 42 and 8 subfamilies respectively inP. pusilliflora. Furthermore, 81 MADS-box genes were identified inP. pusilliflora, accompanying expansions of the SVP and AGL15 subfamilies and loss of the TM3 subfamily. Our assembly of a high-qualityP. pusillifloragenome will be valuable for further research on cherries and molecular breeding.