The corticospinal tract (CST) facilitates skilled, precise movements, which necessitates that corticospinal neurons (CSN) establish segmentally specific connectivity with spinal circuitry. Recent work has identified CSN-intrinsic molecular determinants that regulate CSN axonal projections to specific segmental levels of the neuraxis and that these are expressed by CSN subpopulations that project to distinct levels of the neuraxis. In the present study, we establish the use of a newly generated Klhl14-T2A-Cre knock-in reporter mouse line to investigate axonal projections of a specific CSN subpopulation, CSNBC-lat. Breeding these mice with reporter mice shows recombination across multiple cell types and did not recapitulate the known specificity of Klhl14 expression by CSNBC-lat. However, by using conditional anterograde and retrograde labeling during development, we identify this specificity of Cre expression. We establish AAV-mediated gene delivery in Klhl14-T2A-Cre mice as an approach to reliably analyze CSNBC-lat axon targeting. We also confirm that this approach can be utilized to perform gain-of-function experiments which in turn identifies known regulators of CSN axon targeting – Crim1 and Cbln1. Finally, using intersectional viral labeling, we establish that Klhl14+ CSNBC-lat show topographical specificity of axonal projections within the brainstem. Our results establish this strategy as a novel approach for in vivo testing of candidate gene function in controlling context-appropriate CSN axon guidance in a relatively high-throughput manner.