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Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause phenotypic changes in the glands upon knock-down, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. Gene ontology analysis revealed that many of those genes are encoding enzymes (peptidases and cytochromes P450) as well as proteins involved in membrane trafficking with an enrichment in lysosome and mineral absorption pathways. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones in the gland reservoirs, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. However, only one out of eight genes identified by a transcriptomics approach known to cause phenotypic changes of the glands upon knock-down was recognized by this phenotypic screen, indicating the limitation of such a non-redundant first-pass screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.