Plethodontid salamanders have served as an informative vertebrate system for studying the role of chemical signals in facilitating social and reproductive behaviors. Individuals produce complex mixtures of chemicals from multiple glandular regions. In total, these secretions convey a wide variety of information, and are important for numerous inter- and intraspecific interactions. In order for a signal to convey complex information, it must vary in its composition. This research explores additional aspects of pheromone variation, within and between species, and discusses how this variation may affect the many functions of chemical signals in these species. Chapter One provides an overview of communication, chemical signaling, and essential background for the system in study. Chapter Two documents the degree of intraspecific variation in a major courtship pheromone protein, Plethodontid Receptivity Factor (PRF). Chapter Three explores the evolution of an additional level of signal complexity (post-translational modification via glycosylation) in the same pheromone, and documents substantial inter- and intraspecific variation in this trait. Lastly, Chapter Four uses high-throughput RNA sequencing to provide the first detailed description of the genes expressed by three other signaling glands: the cloacal glands, postcloacal gland, and the dorsal tail base. These tissues are involved in different aspects of reproductive, territorial, and other social behavior. Finally, Chapter Five summarizes the findings of this dissertation, incorporates these findings into an integrated model of chemical communication in plethodontids, and discusses key hypotheses to consider and directions for future research.