The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.