Headache is a common complaint after mild traumatic brain injury (mTBI). Changes in the CNS lipidome were previously associated with acrolein-induced headache in rodents. mTBI caused similar headache-like symptoms in rats; therefore, we tested the hypothesis that mTBI might likewise alter the lipidome. Using a stereotaxic impactor, rats were given either a single mTBI or a series of 4 mTBIs 48 h apart. 72 h later for single mTBI and 7 days later for repeated mTBI, the trigeminal ganglia (TG), trigeminal nucleus (TNC), and cerebellum (CER) were isolated. Using HPLC/MS/MS, ~80 lipids were measured in each tissue and compared to sham controls. mTBI drove widespread alterations in lipid levels. Single mTBI increased arachidonic acid and repeated mTBI increased prostaglandins in all 3 tissue types. mTBI affected multiple TRPV agonists, including N-arachidonoyl ethanolamine (AEA), which increased in the TNC and CER after single mTBI. After repeated mTBI, AEA increased in the TG, but decreased in the TNC. Common to all tissue types in single and repeated mTBI was an increase the AEA metabolite, N-arachidonoyl glycine, a potent activator of microglial migration. Changes in the CNS lipidome associated with mTBI likely play a role in headache and in long-term neurodegenerative effects of repeated mTBI.