The Jamaican click beetle Pyrophorus plagiophthalamus (Coleoptera: Elateridae) is unique among all bioluminescent organisms in displaying a striking light color polymorphism [Biggley, W. H., Lloyd, J. E. & Seliger, H. H. (1967) J. Gen. Physiol. 50, 1681-1692]. Beetles on the island vary in the color of their ventral light organs from yellow-green to orange and their dorsal organs from green to yellow-green. The genetic basis for the color variation involves specific amino acid substitutions in the enzyme luciferase. Here, we show that dorsal and ventral light color in P. plagiophthalamus are under separate genetic control, we resolve the allelic basis for color variation, and, through analyses of luciferase sequence variation, we demonstrate that natural selection has produced a long-term adaptive trend for longer wavelength (more orange) ventral light on Jamaica. Our results constitute a novel example connecting the selective fixation of specific nucleotides in nature to their precisely determined phenotypic effects. We also present evidence suggesting that a recently derived ventral orange luciferase allele on the island has deterministically increased in frequency. Thus, the current luciferase polymorphism for P. plagiophthalamus appears to be mirroring the long-term anagenic trend on Jamaica, revealing a possible ongoing adaptive color transition in progress.