A method was developed to characterize the genetic heterogeneity of the chalcone synthase gene intron within the Petunia integrifolia (Hook.) Schinz & Thell. species complex. The DNA from wild species collected from known locations was used to amplify the chalcone synthase gene intron through the polymerase chain reaction (PCR). The resulting PCR product was then characterized by Rsa 1 restriction, revealing a degree of heterogeneity that could be used to characterize the species genetically. Of the four different species that were characterized, two could be placed in the same genetic grouping. This study shows that the variation in the intron of the Chs A gene may be species-specific.