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Abstract Over the past decades, many critical molecular players have been uncovered to control distinct steps in olfactory circuit assembly in Drosophila. Among these, multi-member gene families of cell surface proteins are of interest because they can act as neuron-specific identification/recognition tags in combinations and contribute to circuit assembly in complex brains through their heterophilic or homophilic interactions. Recently, a multi-protein interactome has been described between the Beat and Side families of IgSF proteins. Here, we use the publicly available single-cell RNA-seq datasets and newly generated gene trap transgenic driver lines to probe thein vivospatial expression pattern of thebeat/sidegene families in odorant receptor neurons (ORNs) and their synaptic target projection neurons (PNs).Our results revealed that each ORN and its synaptic target PN class expresses a class-specific combination ofbeat/sidegenes, hierarchically regulated by lineage-specific genetic programs. Though ORNs or PNs from closer lineages tend to possess more similarbeat/sideprofiles, we also found many examples of divergence from this pattern among closely related ORNs and closely related PNs. To explore whether the class-specific combination ofbeats/sidesdefines ORN-PN matching specificity, we perturbed presynapticbeat-IIaand postsynapticside-IVin two ORN-PN partners. However, disruption of Beat-IIa-Side-IV interaction did not produce any significant mistargeting in these two examined glomeruli. Though without affecting general glomerular targeting, knockdown ofsidein ORNs leads to the reduction of synaptic development. Interestingly, we found conserved expression patterns ofbeat/sideorthologs across ORNs in ants and mosquitoes, indicating the shared regulatory strategies specifying the expression of these duplicated paralogs in insect evolution. Overall, this comprehensive analysis of expression patterns lays a foundation for in-depth functional investigations into how Beat/Side combinatorial expression contributes to olfactory circuit assembly.