The evolutionary mechanisms responsible for the loss of eyes in cave animals are still unresolved. Hypotheses invoking natural selection or neutral mutation have been advanced to explain eye regression. Here we describe comparative molecular and developmental studies in the teleost Astyanax mexicanus that shed new light on this problem. A. mexicanus is a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish) from different caves. We first review the evolutionary relationships of Astyanax cavefish populations and conclude that eye degeneration may have evolved multiple times. We then compare the mechanisms of eye degeneration in different cavefish populations. We describe the results of experiments showing that programmed cell death of the lens plays a key role in controlling eye degeneration in these cavefish populations. We also show that Pax6 gene expression and fate determination in the optic primordia are modified similarly in different cavefish populations, probably due to hyperactive midline signaling. We discuss the contributions of the comparative developmental approach toward resolving the evolutionary mechanisms of eye degeneration. A new hypothesis is presented in which both natural selection and neutral mutation are proposed to have roles in cavefish eye degeneration.