Relationships among North American plethodontid salamanders have been the subject of numerous molecular studies. In particular, immunological investigations of albumin evolution using the quantitative technique of micro-complement fixation have provided insights into relationships between the genera Ensatina and Plethodon, as well as within Plethodon. However, examination of relationships within eastern Plethodon has been hindered due to lack of antisera from a representative of each species group. An antiserum to albumin from P. wehrlei (wehrlei group) was produced and compared to all available Plethodon antigens. With this antiserum, all currently recognized species groups of eastern Plethodon (glutinosus, cinereus, welleri, and wehrlei) are now represented in a reciprocal matrix of immunological distance values. Phylogenetic trees derived from these data indicate that the welleri and glutinosus groups are sister taxa, and cluster with the wehrlei group; the cinereus group is the most distant within the eastern Plethodon. This topology conflicts with that based on electrophoretic and morphological data, which suggest that the glutinosus and wehrlei groups are sister taxa. A close examination of the albumin immunological data shows that the rate of albumin evolution within eastern Plethodon has been variable, and therefore these data may be less reliable for phylogenetic reconstruction in this taxon.