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, RhB) in five different cone-like photoreceptor types 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 consititutes a natural experiment in which to study the selection pressures underlying opsin gene expression and the evolution of colour discrimination. By analysing 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 nm 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 substiution (Ser to Pro) at site 164 is responsible for a blue-shift of 19 nm of the LWS visual pigment of P. marinus compared to 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 colour vision and that the complement and tuning of these visual pigments reflect the ecology and light environment of these phylogenetically basal vertebrates.