Deficits in sensory and motor function after spinal cord injury are attributable primarily to the interruption of long sensory and motor axonal tracts in the spinal cord. Different spinal cord tracts display different vulnerability to the injury, capability to regenerate, and contribution to functional recovery. Due to the complexity of the central nervous system (CNS) tracts, it is worthwhile to exploit how to exquisitely label them within the spinal cord and examine their connections to the brain or periphery. Retrograde axonal tract tracing technologies are powerful tools for identifying axonal connections in the normal or injured spinal cord. With appropriate injury models and retrograde tracing techniques to transport tracers from axon terminal to the soma, neuronal origin of targeted axons can be determined. After tracing, tissue is collected following appropriate survival time. To detect tracers inside the neuronal soma, nucleus, and corresponding dendrites, various labeling techniques are used which include direct fluorescence, immunohistochemistry, immunofluorescence, and autoradiography. When combined with anterograde tracing techniques, retrograde tracers may be used to establish anatomical reorganizations of neural networks. When combined with immunohistochemistry, retrograde tracing may be used for neurochemical characterization of specific neuronal pathways or specific cell types.