Male sterility in peach (Prunus persica L.), characterized by the absence of fertile pollen grains in the anther, is determined by a recessive allele in homozygosis of the major gene located on chromosome 6. Developing tightly linked molecular markers can help identify appropriate peach parents or male-sterile plants for early culling in segregating progenies, thereby increasing breeding efficiency. In this study, we performed comprehensive research integrating genome-wide association study, bulked segregant analysis, and tissue-specific transcriptome sequencing for precisely characterizing the genes associated with male sterility and fertility in peach. We identified the candidate gene Prupe.6G027000, which encodes an ATP-binding cassette transporter G family member 26 (ABCG26), as a reliable candidate for controlling the targeted traits, as indicated by gene expression profiling and validated by quantitative real-time polymerase chain reaction, in situ hybridization, and virus-induced gene silencing. Prupe.6G027000 was transcribed preferentially on the tapetum and microspore surface, and its transient silencing caused severe pollen abortion in peach. The genotypes of nonsynonymous single-nucleotide variation (T >
C) harbored in the coding region of Prupe.6G027000 exhibited approximately 96.2% consistency with male fertile or sterile phenotype in 579 peach accessions. These findings lay the foundation for dissecting the genetic basis of male fertility traits, and facilitating the establishment of a marker-assisted selection system in peaches.