A broad survey of immunological affinities among squirrels (Sciuridae) was achieved by testing sera of 36 species in immunodiffusion using antisera to the sera of 11 species. The results (net spur values) were summarized using set theoretical notation in a computer program which ordered the compared species in order of their divergence from the homologous species. A dendrogram was constructed by the Unweighted Pair Group Method. Species within genera were usually discriminated by this method and taxa recognized on morphological evidence as related were more similar in their proteins than unrelated ones. Among members of the Sciuridae the first branching was of chipmunk-ground squirrel and tree squirrel-flying squirrel lineages. The immunological evidence for the divergence of chipmunks and ground squirrels early in the evolutionary history of the family is in accord with the fossil record. Systematic relationships among ground squirrels suggested by the comparison data correlate with accepted classification; prelimmary data also support the hypothesis that prairie dogs are most closely related to the genus Spermophilus. The data suggest divergence of three phyletic lines of tree squirrels and two of flying squirrels. The Asian tree squirrel lineage includes members of the tribe Callosciurini (Callosciurus, Sundasciurus, Tamiops, and Menetes) and the Indian Funambulus which were more similar to each other antigenically than to any other tree squirrels. The giant squirrel genus Ratufa represents a separate phyletic line and Sciurus and Tamiasciurus constitutes a third line. Except for the Asian Iomys, all flying squirrels examined (Glaucomys, Petinomys, Pteromys, and Petaurista) were more closely related to each other than to other squirrels, and as a group show a close affinity to the Sciurus phyletic line. This observation opposes the present classification which assigns flying squirrels to the subfamily Petauristinae and the tree squirrels and ground squirrels to the subfamily Sciurmae. The immunodiffusion technique permits comparisons over a wide taxonomic range, providing data useful at several taxonomic levels; Aplodontia rufa, the most primitive living rodent, was greatly divergent from all squirrels.