The divergent evolution of niche-related traits can facilitate adaptive radiation, yet identification of the genetic or molecular mechanisms underlying such trait changes remains a major challenge in evolutionary biology. Conducting a detailed morphological comparison along growth trajectories is a powerful method for observing the formation of differences in niche-related traits. Here, we focused on hindlimb length of Anolis lizards, differences in which are related to adaptation for use of different microhabitats. We measured the length of hindlimb skeletons in different ecomorphs of anole lizards (A. sagrei, a trunk-ground ecomorph with long hindlimbs, and A. angusticeps, a twig ecomorph with short hindlimbs) from early embryonic stages to adulthood, to determine which hindlimb elements mainly differentiate the species and the timing of the formation of these differences. With respect to the digit, differences between the species mainly occurred during the embryonic stages of interdigit reduction, when the cartilage of the distal phalanges was simultaneously forming. In addition, we compared the relative length of developing autopods in early embryonic stages using whole-mount in situ hybridization before the formation of the cartilaginous bones, and the results showed that the relative growth rate of the Hoxa11-negative distal region in A. sagrei was greater than that in A. angusticeps. Our results show that there are several important developmental stages for hindlimb length differentiation between A. angusticeps and A. sagrei, depending on which hindlimb element is considered. In particular, the species differences were largely due to variations in digit length, which arose at early embryonic stages.