Protein pheromones in salamanders of the genus Plethodon have experienced rapid and pervasive directional selection. Variation in mate recognition components, such as the sex-specific pheromones used by plethodontid salamanders, may influence sexual incompatibility and therefore provide a biochemical mechanism for the maintenance of discrete, isolated populations. Recent studies suggest that multiple, genetically distinct lineages of Eastern Red-Backed Salamanders (Plethodon cinereus) are present throughout their broad range. Representative populations from two of these lineages (the Ohio [OH] and Pennsylvania [PA] clades) span the southern shore of Lake Erie in northern Ohio. This distribution pattern creates a unique opportunity to study how phenotypic differences may reinforce population boundaries and possibly lead to speciation. The objectives of this study were to 1) characterize the pheromone profiles of male P. cinereus and 2) determine pheromone variation among populations and between the OH and PA clades. The composition of proteins associated with two known courtship pheromones, Plethodontid Modulating Factor (PMF) and Plethodontid Receptivity Factor (PRF), were compared among eight populations in northern Ohio. Analyses of Similarity (ANOSIM) suggest that both PMF and PRF profiles differ among populations but not between clades. These data suggest that the sex-specific pheromones of P. cinereus in the two clades are not yet different enough to allow reproductive isolation between the two genetic lineages. Although the relative roles of selection and genetic drift are unknown in our populations, specific pheromone isoforms and their effects on mate compatibility should be the focus of future studies aiming to determine mechanisms involved in maintaining population differences.