Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases.