To clarify the polyploid origin of the sweetpotato, we analyzed retentions of three distinctive types of Waxy intron 2 (Wx-In2) variants among 27 sweetpotato lines and 24 selected relatives and their phylogenetic relationships with Wx-In2 from 11 closest relatives. The three types of Wx-In2 effectively distinguish three diploid constituent genomes of very close homeology in the sweetpotato: Type I is characteristic of some loci in Genome I and III, and Types II and III are specific to loci in Genome II and III. The Type I Wx-In2 variation was found to be retained in 19 sweetpotato lines, Ipomoea littoralis Blume (49), I. tabascana (49), and I. tenuissima (29); Type II to be retained in all 27 sweetpotato lines, I. littoralis Blume, and two I. trifida accessions; Type III to be retained in 13 sweetpotato lines, I. tenuissima, and four distantly related species. Because of the nature of independent random divergence of orthologous intronic sequences, these highly selective retentions of genome-specific or characteristic sweetpotato Wx-In2 variations among four diploid or tetraploid sweetpotato relatives are consistent only with separate lineages of diploid genomes of the sweetpotato. Such an allohexaploid origin of the sweetpotato probably occurred via hybridization between I. tenuissima and I. littoralis Blume, derived earlier from I. trifida and an unidentified species sibling to I. tenuissima. However, neither the involvement of I. tabascana nor a multiple origin of the sweetpotato can be ruled out. The inference is supported by maximal likelihood relationships between the three types of Wx-In2 from the sweetpotato and Wx-In2 from its 11 closest relatives.