Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC 220.127.116.11; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a approximately 50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChEs. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti-regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene+azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species-specific AChE ligands that conform to the determinants in the gorge.