The genetic distance for three Petunia species was determined based upon an intron in the chalcone synthase gene. The sequence of the intron was obtained for P. integriflolia ssp. integriflolia var. depauperata, P. integriflolia ssp. integriflolia Torres ecotype, P. altiplana and P. littoralis. These species are very closely related and believed by some taxonomists to be part of a large single species complex. In all the taxa, the intron contained multiple repeated and inverted sequences. The P. integriflolia ssp. integriflolia Torres ecotype intron differed from the P. integriflolia subsp. integriflolia var. depauperata intron in 3 of 930 nucleotides. While, the P. littoralis intron differed from the P. integrifolia subsp. integrifolia var. depauperata intron in 15 of 930 nucleotides. As compared to the P. integrifolia subsp. integriflolia var. depauperata intron, the intron in P. altiplana intron was longer (1125 bp), had a section of 338 nucleotides with a completely different sequence, and differed by 27 of 787 nucleotides in the common sequence.