The agnathans (lampreys and hagfishes) are representatives of the jawless fishes and constitute the first lineage of extant vertebrates to evolve within chordate phylogenetic history. Previously, we showed that the southern hemisphere pouched lamprey Geotria australis has the potential for pentachromacy with the expression of five visual pigment (opsin) genes (LWS, SWS1, SWS2, RhA, and RhB) in five different cone-like photoreceptors for life in a brightly lit environment exposed to a broad spectrum of light. In contrast, the northern hemisphere sea lamprey Petromyzon marinus dwells in a wide range of depths that are relatively deeper than the epipelagic waters inhabited by G. australis. Thus, the light levels of the habitat in which the sea lamprey resides are greatly diminished and different regions of the light spectrum are differentially absorbed. Therefore, the visual systems of these two species of lamprey constitute a natural experiment in which to study the selection pressures underlying opsin gene expression and the evolution of color discrimination. By analyzing the opsin genes of P. marinus, we show the expression of two intact retinal opsins, RhA and LWS, which, when regenerated with 11-cis retinal, give peak spectral sensitivities (lambda(max) values) of 501 and 536 nm, respectively. In contrast to G. australis, the genome of P. marinus possesses remnants of SWS1 and SWS2 pseudogenes, which with the loss of RhB, suggests that P. marinus is a dichromat. Using site-directed mutagenesis, we show that a single amino acid substitution (Ser to Pro) at site 164 is responsible for a blue shift of 19 nm of the LWS visual pigment of P. marinus compared with G. australis, which may reflect habitat differences between the two species. Based on these studies, we propose that gene loss (or duplication) and subsequent mutation plays an important role in the evolution of color vision and that the complement and tuning of these visual pigments reflect the ecology and light environment of these phylogenetically basal vertebrates.