This is a systematic study of three externally similar species of plethodontid salamanders (genus Desmognathus) previously considered to occur sympatrically in northern Florida (D. auriculatus, D. brimleyorum, and D. fuscus). Larval morphology, color pattern, relative size, and tail morphology were reinvestigated. The morphology of teeth, jaw profile, premaxillary fontanelle, and prearticular spine were studied in detail for the first time, and color changes due to ontogenetic melanization and to metachrosis were also evaluated. All the above characteristics were found to be taxonomically signifcant. In addition, the microhabitat selection and ecological associates of D. auriculatus and D. fuscus were different, indicating ecological isola-tion of the two species in the area of sympatric contact in northern Florida. Desmognathus brimleyorum Stejneger is a species endemic to the Ouachita Mountains of Arkansas and Oklahoma. The name D. fuscus carri Neill is a synonym of D. auriculatus Holbrook. Populations referred to D. fuscus in this study compared morphologically more closely with D. ochrophaeus from the southern Appalachians than with populations from near the type locality of D. fuscus Rafinesque.