Motor neuron damage and cortical spinal tract (CST) degeneration are pathological features of amyotrophic lateral sclerosis (ALS). We combined whole-brain diffusion tensor imaging (DTI) and three-dimensional magnetic resonance spectroscopic imaging (MRSI) to study the CST at different locations. Eight ALS patients were compared with normal controls. Fractional anisotropy (FA) and mean diffusivity (MD), and the ratio of N-acetyl-aspartate (NAA) to creatine (Cr) were measured at various locations in the CST, including the subcortical white matter (SWM), centrum semiovale (CS), periventricular white matter (PV), posterior limb of the internal capsule (PIC) and cerebral peduncle (CP). Patients showed significantly lower FA than controls in the CST, including the SWM, CS, PV and PIC. Although there was a trend towards elevated MD in ALS patients, this did not reach statistical significance. NAA/Cr ratios were also decreased in ALS patients compared with normal controls, with significant differences in the SWM and PV but not in PIC. Combined whole-brain DTI and MRSI can detect axonal degeneration in ALS. Measurements of FA obtained in the SWM, CS, PV and PIC, and NAA/Cr ratios in the SWM and PV yield the most robust results.