Although turtles have been generally grouped with the most primitive reptile species, the origin and phylogenetic relationships of turtles have remained unresolved to date. To confirm the phylogenetic position of turtles in amniotes, we have cloned and determined the cDNA sequences encoding for skink lactate dehydrogenase (LDH)-A and LDH-B, snake LDH-A, and African clawed frog LDH-A; four alpha-enolase cDNA sequences from turtle, alligator, skink, and snake were also cloned and determined. All of these eight cDNA sequences, as well as the previously published LDH-A, LDH-B, and alpha-enolase of mammals, birds, reptiles, and African clawed frog, were analyzed by the phylogenetic tree reconstruction methods of neighbor-joining, maximum parsimony, and maximum likelihood. In the phylogenetic analyses, the turtle was found to be closely related to the alligator. Also, we found that the turtle had diverged after the divergence of squamates and birds. This departs from previous hypotheses of turtle evolution and further suggests that turtles are the latest of divergent reptiles, having been derived from an ancestor of crocodilian lineage within the last 200 million years.