MRD57 (or autosomal dominant 57) is a rare neurodevelopmental disorder linked to genetic mutations in the serine/threonine kinase, TLK2. MRD57 is characterised by TLK2 haploinsufficiency and in some cases, its loss or impaired kinase function. TLK2 is an established cell cycle regulator, and in contrast to MRD57, cancers upregulate TLK2 activity to drive tumour growth. MRD57 patients have a range of clinical phenotypes including delayed psychomotor development, language delay, hypotonia, gastrointestinal issues, dysmorphic facial features and global developmental delay. However, the neuronal functions of TLK2 are poorly understood. We aimed to gain mechanistic insight into how TLK2 mutations cause MRD57 by determining where TLK2 is expressed in the brain and its role in neuronal differentiation. In mouse brain cDNA and human brain transcriptomic data, we identified splice variant diversity in the N-terminus of TLK2 in which its nuclear localisation sequence (NLS) is located. Using splice-specific in situ hybridisation probes, we demonstrated expression of TLK2 isoforms in the mouse hippocampus and cerebellum that contain and lack the NLS. Subcellular fractionation and immunostaining confirmed nuclear and non-nuclear localisation of TLK2 in differentiated human neuroblastoma cells. Furthermore, during neuronal differentiation the expression profile of TLK2 splice variants changes, together with an increase of non-nuclear TLK2. Our data highlight the need to establish the non-nuclear neuronal substrates of TLK2 and how the loss of TLK2 activity in MRD57 might impact on their function in neuronal development.