The isolation of disease resistance genes introduced from wild or related cultivated species is essential for understanding their mechanisms, spectrum and risk of breakdown. To identify target genes not included in reference genomes, genomic sequences with the target locus must be reconstructed. However,de novoassembly approaches of the entire genome, such as those used for constructing reference genomes, are complicated in higher plants. Moreover, in the autotetraploid potato, the heterozygous regions and repetitive structures located around disease resistance gene clusters fragment the genomes into short contigs, making it challenging to identify resistance genes. In this study, we report that ade novoassembly approach of a target gene-specific homozygous dihaploid developed through haploid induction was suitable for gene isolation in potatoes using the potato virus Y resistance geneRychcas a model. The assembled contig containingRychc-linked markers was 3.3 Mb in length and could be joined with gene location information from the fine mapping analysis.Rychcwas successfully identified in a repeated island located on the distal end of the long arm of chromosome 9 as a Toll/interleukin-1 receptor-nucleotide-binding site-leucine rich repeat (TIR-NBS-LRR) type resistance gene. This approach will be practical for other gene isolation projects in potatoes.