Studies of molecular variation within the chloroplast genome have been used to determine phylogenetic relationships within the evolutionarily complex family Cactaceae. Changes detected in the chloroplast DNA (cpDNA) involving structural rearrangements (inversions, deletions, etc.), restriction site variation, and gene sequence variation have provided phylogenetically informative data, useful in the construction of cladograms which infer evolutionary relatedness among the various cactus groups investigated. Examples are provided from studies of representative cacti for each of these types of detected cpDNA variation. Applicability of these methods to resolving fundamental questions in cactus evolution and systematics are also discussed.