Pheromones are important sexual signals in most animals, but research into their evolution is largely biased toward insects. Lampreys are a jawless fish with a relatively well-understood pheromone communication system and offer a useful opportunity to study pheromone evolution in a vertebrate. Once sexually mature, male sea lamprey (Petromyzon marinus), and likely other lampreys, produce and release bile acids that act as sex pheromones. Spawning males do not feed and therefore produce bile acids primarily for sexual communication whereas larvae produce the same bile acids but for digestion, offering an opportunity to compare the evolution of bile acids produced for sexual versus nonsexual functions. We profiled eight pheromone-related bile acids in livers from larvae and males and determined the effect of life stage on intra- and interspecific variation in bile acid production. Our results indicate less variation among males than larvae within P. marinus but more variation among species for males than larvae. We postulate that bile acid production in males is shaped by directional or stabilizing selection that reduces variance within P. marinus and directional or disruptive selection that promotes diversification across species. Although our results offer support for a role of sexual selection in the evolution of a lamprey pheromones, they do not eliminate possible roles of other aspects of lamprey ecology.