The genetic mechanisms underlying regressive evolution—the degeneration or loss of a derived trait—are largely unknown, particularly for complex structures such as eyes in cave organisms. In several eyeless animals, the visual photoreceptor rhodopsin appeared to retain functional amino-acid sequences. Hypotheses to explain apparent maintenance of function include weak selection for retention of light-sensing abilities and its pleiotropic roles in circadian rhythms and thermotaxis. In contrast, we show that there has been repeated loss of functional constraint of rhodopsin in amblyopsid cavefishes, as at least three cave lineages have independently accumulated unique loss-of-function mutations over the last 10.3 My. While several cave lineag...