Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFP-labeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.