Lampreys are one of the two surviving groups of jawless vertebrates, whose ancestors arose more than 540 million years ago. Some species, such as Geotria australis, are anadromous, commencing life as ammocoetes in rivers, migrating downstream to the sea, and migrating back into rivers to spawn. Five photoreceptor types and five retinal cone opsin genes (LWS, SWS1, SWS2, RhA, and RhB) have previously been identified in G. australis. This implies that the ancestral vertebrates possessed photopic or cone-based vision with the potential for pentachromacy. Changes in the morphology of photoreceptors and their spectral sensitivity are encountered during differing aquatic phases of the lamprey lifecycle. To understand the molecular basis for these changes, we characterized the visual pigments and measured the relative levels of opsin expression over two lifecycle phases that are accompanied by contrasting ambient light environments. By expressing recombinant opsins in vitro, we show that SWS1, SWS2, RhA, and RhB visual pigments possess lambda(max) values of 359, 439, 497, and 492 nm respectively. For the LWS visual pigment, we predict a lambda(max) value of 560 nm based on key spectral tuning sites in other vertebrate LWS opsins. Quantitative reverse transcriptase-polymerase chain reaction reveals that the retinal opsin genes of G. australis are differentially regulated such that the visual system switches from a broad sensitivity across a wide spectral range to a much narrower sensitivity centered around 490-500 nm on transition from marine to riverine conditions. These quantitative changes in visual pigment expression throughout the lifecycle may directly result from changes in the lighting conditions of the surrounding milieu.