A 3D chaos game is shown to be a useful way for encoding DNA sequences. Since matching subsequences in DNA converge in space in 3D chaos game encoding, a DNA sequence’s 3D chaos game representation can be used to compare DNA sequences without prior alignment and without truncating or padding any of the sequences. Two proposed methods inspired by shape-similarity comparison techniques show that this form of encoding can perform as well as alignment-based techniques for building phylogenetic trees. The first method uses the volume overlap of intersecting spheres and the second uses shape signatures by summarizing the coordinates, oriented angles, and oriented distances of the 3D chaos game trajectory. The methods are tested using: (1) the first exon of the beta-globin gene for 11 species, (2) mitochondrial DNA from four groups of primates, and (3) a set of synthetic DNA sequences. Simulations show that the proposed methods produce distances that reflect the number of mutation events; additionally, on average, distances resulting from deletion mutations are comparable to those produced by substitution mutations.