To investigate the relationship of glutamate and glutamate transporter expression in human gliomas and surrounding peritumoral brain to the presence of tumor-associated seizures (TAS). We studied a retrospective (group 1: 190 patients) and then a prospective (group 2: 98 patients) cohort of patients who underwent a craniotomy for a supratentorial glioma. Tumor and peritumor tissue specimens were assayed for glutamate concentration and expression of glial glutamate transporters. Differences between the seizure (TAS) and seizure-free (non-TAS) groups were compared. A total of 42% of patients had TAS, with 95% of seizures first occurring preoperatively. Clinical factors independently associated with risk of TAS were younger age, temporal lobe location, and tumors with oligodendroglial components. Molecular features in tumor specimens associated with TAS were higher glutamate concentrations, reduced EAAT2 expression, and increased system X(c)(-) expression. In group 2, these results were also replicated in the peritumor tissue. Logistic regression analysis identified raised glutamate concentrations in tumor and peritumor tissue, increased expression of peritumor system X(c)(-), younger age, temporal lobe location, and tumors with oligodendroglial components as independently predictive of preoperative seizures. Relative increased glutamate concentration in gliomas, and altered glutamate transporter expression, are associated with the presence of TAS and may play a mechanistic role in their pathogenesis.