The platypus is a venomous monotreme. Male platypus possesses a spur on their hind legs connected to glands in the pelvic region. They only produce venom during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified include antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.