Nucleotide sequence data from a 556 bp segment of the mitochondrial 16S ribosomal RNA gene support the partition of emydid turtles into two distinct subfamilies. Phylogenetic analysis identifies two major clades and corroborates previous morphological assignments of genera to the subfamilies Deirochelyinae and Emydinae. Within the subfamily Deirochelyinae, Deirochelys appears to be the sister taxon to all other genera in the subfamily, and support is found for a clade that includes Trachemys, Graptemys, and Malaclemys. However, the other generic relationships within this subfamily are not well resolved. Within the Emydinae, the genus Terrapene is monophyletic. Conversely, the genus Clemmys is paraphyletic, necessitating a new generic arrangement of the species now considered to be in Clemmys, Emydoidea, and Emys.