Re-domestication of diploid potato (Solanum tuberosum) into a seed crop is an innovative breeding method to accelerate genetic improvement. Seed propagation would allow hybrid production and mix superior alleles. However, almost all diploid potatoes in nature are self-incompatible (SI). Gametophytic self-incompatible (GSI) is a widespread SI in Solanaceae and is controlled by the S locus that contains a ribonuclease (S-RNase) and multiple F-box (SLFs); however, the genetic diversity of the S locus in potato is unclear. This study identified 21 S-RNase alleles involved in SI from 194 diploid potato accessions by large-scale transcriptome sequencing. The levels of amino acid similarity among different S-RNase proteins varied from 31.3 to 95.8%. S2 allele is the most widespread in 194 diploid potatoes and is mainly distributed in the S. tuberosum Group Phureja. Based on genomic annotation and expression analysis, we identified 12 potential functional SI male-determinant genes, S-locus F-box (SLFs), encoding F-box proteins in the S2 locus on a genomic region of approximately 13 Mb. Comparative genomics analysis showed that eight SLF genes are relatively conserved among four homozygous S locus. The Ka and Ks analysis suggested that S-RNase and intra-haplotypic SLF genes have co-evolved. These findings help select suitable pollinators, combine more hybrid combinations, and fully use heterosis to accelerate diploid potato breeding.