The pharmacological agent nitroglycerin (NTG) elicits hyperalgesia and allodynia in mice. This model has been used to study the neurological disorder of trigeminovascular pain or migraine, a debilitating form of hyperalgesia. The present study validates hyperalgesia in an established mouse model of chronic migraine triggered by NTG and advances the understanding of the associated molecular mechanisms. The RNA-seq profiles of two nervous system regions associated with pain, the trigeminal ganglia (TG) and the nucleus accumbens (NAc), were compared in mice receiving chronic NTG treatment relative to control (CON) mice. Among the 109 genes that exhibited an NTG treatment-by-region interaction, solute carrier family 32 (GABA vesicular transporter) member 1 () and preproenkephalin ) exhibited reversal of expression patterns between the NTG and CON groups. Erb-b2 receptor tyrosine kinase 4 () and solute carrier family 1 (glial high affinity glutamate transporter) member 2 () exhibited consistent differential expression between treatments across regions albeit at different magnitude. Period circadian clock 1 () was among the 165 genes that exhibited significant NTG treatment effect. Biological processes disrupted by NTG in a region-specific manner included adaptive and innate immune responses; whereas glutamatergic and dopaminergic synapses and rhythmic process were disrupted in both regions. Regulatory network reconstruction highlighted the widespread role of several transcription factors (including , and ) among the NTG-disrupted target genes. These results advance the understanding of the molecular mechanisms of hyperalgesia that can be applied to therapies to ameliorate chronic pain and migraine.