A fragment of the Pain-1 vacuolar invertase locus was sequenced and its structure and polymorphism were characterized in 17 species of the genus Solanum, including the subgenera Potatoe, Solanum, Leptostemonum, Minon, and Brevantherum. The fragment size varied from 603 to 977 bp as a result of multiple indels in the region of intron III. A total of 80 single nucleotide polymorphisms were found in the coding region, of which 34 caused amino acid substitution in the protein product. Several substitutions and indels were specific to individual taxons or taxon groups, including potato and tomato species. The genetic distances and phylogenetic trees obtained supported the commonly accepted taxonomic classification of the species, indicating that the Pain-1 fragment is suitable for taxonomic identification and phylogenetic studies in Solanaceae.