Many species of plethodontid salamanders exist as complexes of genetically fragmented, parapatrically and allopatrically distributed groups of populations. A complex describes a group of cryptic species with adjacent or slightly overlapping distributions. These complexes provide a unique opportunity to examine the mode and tempo of speciation, specifically illuminating the mechanisms that maintain genetic cohesion. My research focuses on the Desmognathus ochrophaeus complex, composed of a number of small plethodontid salamanders inhabiting streams and seeps in the Appalachian Mountains. I have examined the mode and tempo of speciation in this complex using multiple markers to characterize divergence, assess gene flow, and evaluate reproductive isolation. On a broad scale, markers are concordant in recognizing distinct, evolutionary lineages. On a finer scale, however, markers are less condordant, indicating differential gene flow of markers across regions of secondary contact. Data indicate species can show high degrees of genetic divergence and yet are still capable of interbreeding, unlike patterns seen in other organisms. Asymmetry in mating preferences, however, indicates divergence in mate recognition systems has occurred. Furthermore, levels of isolation generally correspond to gene flow inferred from markers, indicating that groups are behaviourally isolated. Phyloethological analysis indicates that loss of some courtship behavior patterns may contribute to reproductive isolation. Molecular and behavioural data indicate species in the Desmognathus ochrophaeus complex probably speciated in allopatry in the Southern Appalachians, sometime during the Pliocene, with some groups experiencing repeated periods of isolation followed by recontact.