L-DOPA-induced dyskinesia (LID) is a major complication of Parkinson’s disease (PD) treatment, yet its underlying neurochemical and molecular mechanisms remain incompletely understood. Here, we employed an integrative spatial omics approach, combining high-resolution mass spectrometry imaging (MSI) of neurotransmitters, metabolites, and lipids, to investigate LID-associated alterations in a non-human primate PD model. Using matrix-assisted laser desorption/ionization (MALDI)-MSI, we spatially mapped neurotransmitters, acetylcholine (ACh) metabolism, and lipid alterations in key motor-related brain regions of MPTP-lesioned macaques with and without LID following chronic L-DOPA treatment. LID was characterized by widespread dysregulation of L-DOPA metabolism, including excessive extrastriatal L-DOPA accumulation and abnormal dopamine turnover, particularly in the primary motor cortex and associative cortical areas. Notably, ACh and its metabolites showed significant changes in the globus pallidus interna (GPi), claustrum, and precentral gyrus, implicating cholinergic dysfunction in LID pathophysiology. These neurotransmitter alterations were strongly associated with distinct lipidomic changes, including depletion of antioxidant plasmalogen phosphatidylcholines (PCs) in the GPi, claustrum, and primary motor cortex, suggesting increased oxidative stress and membrane instability in LID. Additionally, LID was linked to bidirectional alterations in polyunsaturated fatty acid (PUFA)-containing glycerophospholipids (GPLs), potentially affecting neuronal membrane properties and synaptic function. Hydroxylated and non-hydroxylated sulfatides displayed inverse patterns, pointing to peroxisomal dysfunction and compromised myelin integrity as contributing factors in LID pathology. Correlation analyses demonstrated strong associations between ACh and lipid dysregulation and LID severity, underscoring the interplay between cholinergic dysfunction, lipid metabolism, oxidative stress, and neurotransmitter imbalances. Our findings provide novel insights into the complex molecular landscape of LID and highlight lipid- and neurotransmitter-targeted pathways as potential therapeutic strategies to mitigate dyskinesia in PD.