Understanding the relative evolutionary importance of parasites to different host taxa is problematic because the expression of disease and resistance are often confounded by factors such as host age and condition. The antibiotic-producing metapleural glands of ants are a potentially useful exception to this rule because they are a key first-line defense that are fixed in size in adults. Here we conduct a comparative analysis of the size of the gland reservoir across the fungus-growing ants (tribe Attini). Most attines have singly mated queens, but in two derived genera, the leaf-cutting ants, the queens are multiply mated, which is hypothesized to have evolved to improve colony-level disease resistance. We found that, relative to body size, the gland reservoirs of most attines are similar in size but that those of the leaf-cutting ants are significantly larger. In contrast, the size of the reservoir did not relate with the evolutionary transition from lower to higher attines and correlated at most only slightly with colony size. The results thus suggest that the relationship between leaf-cutting ants and their parasites is distinctly different from that for other attine ants, in accord with the hypothesis that multiple mating by queens evolved to improve colony-level disease resistance.