Tract tracing is a fundamental technique in neuroanatomy for examining fiber connections in the nervous system. After the introduction of horseradish peroxidase 40 years ago, many tracing substances have been used for neuroanatomical studies on various nervous systems. Here, we described retrograde tracing techniques using multiple fluorescent tracers, which make it possible to detect axonal collaterals. This technique is useful to study the development of axonal trajectories, as well as regenerative and compensatory mechanisms of animals that undergo neural damage at early stages.