Tomato yellow leaf curl virus (TYLCV) has signifcantly impacted the tomato industry around the world, and the use of insecticides and insect nets have not efectively controlled the spread of this pathogen. The tomato line AVTO1227 is highly resistant to TYLCV. In this study, F2and BC1populations derived from AVTO1227 and the susceptible line Money maker were used to assess the genetic mechanism underlying TYLCV resistance. We have identifed a recessive TYLCV resistance gene, hereby designated as ty-5, which is linked to SlNACI. Genomic DNA pools from resistant and susceptible groups were constructed, and their genomes were resequenced. The ty-5gene was identifed on an interval encompassing the genomic positions 2.22 Mb to 3.19 Mb on tomato chromosome 4. Genotyping using linkage markers further mapped ty-5within the interval between markers ty5–25 and ty5–29, where only the pelotagene is located. Consequently, pelotawas considered as the candidate gene corresponding to ty-5. Two nucleotide transversions within the promoter region and one transversion in exon region of the pelotagene were detected in the parental lines. However, the relative transcript levels of pelotadid not signifcantly difer among the three tomato lines, regardless of TYLCV infection. This study will facilitate marker-assisted breeding for resistance to TYLCV and lay a foundation for the research of the resistance mechanism of ty-5in tomato.