Mutations in GBA1 encoding lysosomal glucocerebrosidase (GCase) are a risk factor for Parkinson’s disease (PD), and reduced GCase function results in imbalances in glycosphingolipid metabolism that accompanies disease pathology. To better understand, glycosphingolipid dysregulation in PD, we developed mass spectrometry imaging approaches to characterise content and distribution of multiple lipid classes in brains from the D409V GBA1 mutant mouse model. Age-matched brains from wild type, D409V heterozygous and homozygous GBA1-mutant mice were analysed using matrix-assisted laser/desorption ionisation (MALDI-MSI) by high-resolution accurate mass spectrometry. Analysis of neutral glycosphingolipids by conventional MALDI-MSI is limited by ionisation efficiency and so adoption of post-ionisation using a second pulsed UV laser (MALDI-2) enables their robust detection. Another technical barrier is anionic sensitivity of gangliosides that we have overcome by incorporation of ammonium sulfate as a matrix dopant in regular MALDI imaging. For both gangliosides and hexosylceramides (containing glucose/galactose), differences in the spatial distribution of specific fatty-acyl chain species across brain regions is evident. MALDI-2 imaging revealed increasing levels of hexosylsphingosine d18:1, a key GCase substrate, within white matter regions correlating with GBA1 mutation status. Implementation of anatomical-driven bioinformatics will further decipher precise changes in the glycosphingolipidome of our clinically relevant PD-mouse model.