The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour de force, the data remain underutilized, partly because of a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioral assays and/or GFP reconstitution across synaptic partners (GRASP) or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch-clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false-positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favorable alternative to GRASP imaging. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotor interneurons they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENT The Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed hundreds of thousands of synaptic connections of the fly larva by manual identification of anatomic landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, providing useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells and show that several genetic driver lines designed to target neurons of the larval connectome exhibit nonspecific and/or unreliable expression.