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Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.