The hyperdiverse ant genus Pheidole, which is characterized by complete dimorphism, shows significant morphometric and behavioral variation between subcastes. In particular, major workers differ in their degree of specialization and repertoire breadth. We studied 3 ecologically different North American Pheidole species that capture the range of variation in the major worker subcaste: P. morrisi (broad repertoire), P. dentata (defense), and P. pilifera (seed milling). Polyethism in all 3 species is also characterized by age-related behavioral development in the minor worker subcaste. We investigated the neural basis of subcaste differences in polyethism using immunohistochemistry. Brains were stained with an antibody against synapsin (SYNORF1). For each individual we measured total brain neuropil volume and the volume of sensory subregions (optic and antennal lobes), and higher integrative subregions (mushroom body calyces, peduncle, and lobes), for young (<3 days) and old (>20 days) major and minor workers of all three species. Within species and subcastes, comparisons among young and old ants revealed age-related increases in the proportional volume of the mushroom bodies. We also constructed a linear discriminant model using 4 size-corrected neuroanatomical variables (the residual volumes of total neuropil, lateral mushroom body calyces, mushroom body peduncles and lobes, and optic lobes) in order to investigate neural variation among species and subcastes in our sample. The model was highly significant (Wilks' L = 0.003, p < 0.0001), and extremely precise, correctly categorizing 100% (35/35) of individuals into the appropriate group (species, age, and subcaste). These results indicate characteristic differences among subcastes and species that are independent of body size. We hypothesize that this neural polymorphism is related to variation in adaptive patterns of division of labor and behavioral development among Pheidole species.