As a model hemimetabolous insect species and an invasive urban pest that is globally distributed, the American cockroach, Periplaneta americana, is of great interest in both basic and applied research. Previous studies on P. americana neuropeptide identification have been based on biochemical isolation and molecular cloning. In the present study, an integrated approach of genomics- and peptidomics-based discovery was performed for neuropeptide identification in this insect species. Using large-scale peptidomic analysis of peptide extracts from 4 different tissues (the central nervous system, corpora cardiac and corpora allata complex, midgut, and male accessory gland), 35 conserved (predicted) and a potential (novel) neuropeptides were then identified. Subsequent experiments revealed the tissue distribution, sex difference, and developmental patterns of 2 conserved neuropeptides (allatostatin B and short neuropeptide F) and a novel neuropeptide PaOGS36577. Our study shows a comprehensive neuropeptidome and detailed spatiotemporal distribution patterns, providing a solid basis for future functional studies of neuropeptides in the American cockroach.