The defensive secretions of anurans contain a large array of chemical compounds that are synthesized in the granular glands and released onto the skin. We used histological and peptidomic analyses to investigate the skin glands and their products in Litoria aurea, Litoria ewingii, and Litoria raniformis, which were introduced to New Zealand from Australia approximately 150 yr ago. The skin glands were induced to release their product by either norepinephrine or electric stimulation. Granular glands in all three species are distributed evenly in both dorsal and ventral skin and share morphological features common in other anurans, such as a contractile myoepithelium that surrounds the syncytial secretory unit. However, differences are observed in the granular ultrastructure between L. ewingii and the more closely related L. aurea and L. raniformis. The latter have larger glands with granules that are opaque and contain homogeneously spaced, diaphanous vesicles, whereas the substructure of the granules in L. ewingii is homogeneous and consists of miniscule vesicles that are either electron opaque or diaphanous. Comparatively large mucous glands in the small-bodied L. ewingii may be attributed to increased mucous requirements due to differences in microhabitat use. Nanospray mass spectrometric analyses confirmed the presence of several unidentified peptides, as well as 11 peptides described previously. Both exposure to norepinephrine and mild electric stimulation of the skin triggered the bulk discharge of gland contents. We discuss potential functional specializations of gland structure and peptide content as mechanisms for predator or pathogen defense.