Motor neuron disease (MND) is a fatal progressive neurodegenerative disorder characterized by the breakdown of the motor system. The clinical spectrum of MND encompasses different phenotypes classified according to the relative involvement of the upper or lower motor neurons (LMN) and the presence of genetic or cognitive alterations, with clear prognostic implications. However, the pathophysiological differences of these phenotypes remain largely unknown. Recently, magnetic resonance imaging (MRI) has been recognized as a helpful MND biomarker. An increasing number of studies is applying advanced neuroimaging techniques in order to elucidate the pathophysiological processes and to identify quantitative outcomes to be used in clinical trials. Diffusion tensor imaging (DTI) is a non-invasive method to detect white matter alterations involving the upper motor neuron and extra-motor white matter tracts. According to this background, the aim of this review is to highlight the key role of MRI and especially DTI, summarizing cross-sectional and longitudinal results of different approaches applied in MND. Current literature suggests that DTI is a promising tool in order to define anatomical "signatures" of the different phenotypes of MND and to track the progressive spread of pathological proteins aggregates.