Background: Cultivated strawberry (Fragaria xananassa Duch.), an allo-octoploid species arising from at least 3 diploid progenitors, poses a challenge for genomic analysis due to its high levels of heterozygosity and the complex nature of its polyploid genome. Results: This study developed the complete haplotype-phased genome sequence from a short-day strawberry, ‘Florida Brilliance’ without parental data, assembling 56 chromosomes from telomere to telomere. This assembly was achieved with high-fidelity long reads and high-throughput chromatic capture sequencing (Hi-C). The centromere core regions and 96,104 genes were annotated using long-read isoform RNA sequencing. Using the high quality of the haplotype-phased reference genome, FaFB1, we identified the causal mutation within the gene encoding Leaf Rust 10 Disease-Resistance Locus Receptor-like Protein Kinase (LRK10) that confers resistance to anthracnose fruit rot (AFR). This disease is caused by the Colletotrichum acutatum species complex and results in significant economic losses in strawberry production. Comparison of resistant and susceptible haplotype assemblies and full-length transcript data revealed a 29-bp insertion at the first exon of the susceptible allele, leading to a premature stop codon and loss of gene function. The functional role of LRK10 in resistance to AFR was validated using a simplified Agrobacterium-based transformation method for transient gene expression analysis in strawberry fruits. Transient knockdown and overexpression of LRK10 in fruit indicate a key role for LRK10 in AFR resistance in strawberry. Conclusions: The FaFB1 assembly along with other resources will be valuable for the discovery of additional candidate genes associated with disease resistance and fruit quality, which will not only advance our understanding of genes and their functions but also facilitate advancements in genome editing in strawberry.