Nondestructive techniques to obtain DNA from organisms can further genetic analyses such as estimating genetic diversity, dispersal, and lifetime fitness, without permanently removing individuals from the population. Possible DNA sources for insects include wing and leg clippings or frass samples. However, these are not feasible approaches for organisms that cannot be removed from their natural environment for long periods or when adverse effects of tissue removal must be avoided. This study evaluated the impacts and efficacy of extracting hemolymph from a defensive secretion to obtain DNA for amplification of microsatellites. A secretion containing hemolymph was obtained from Bolitotherus cornutus (the forked fungus beetle) by perturbation of the defensive gland with a capillary tube. A laboratory experiment demonstrated that the sampling methodology had no impact on mortality, reproductive success, or gland expression. To evaluate the quality of DNA obtained in natural samples, hemolymph was collected from 187 individuals in the field and successfully genotyped at 18 microsatellite loci for 95.7% of samples. These results indicate that hemolymph-rich defensive secretions contain DNA and can be sampled without negative impacts on the health or fitness of individual insects.