Polymorphisms in the glutathione S-transferase (GST) genes, a superfamily that plays a key role in carcinogen metabolism, have been associated with an increased susceptibility to several types of cancer. We wished to evaluate whether variant allelic forms of GST isoenzymes were associated with an increased susceptibility for brain tumors and age of tumor onset. Here, we examined 394 brain tumors (221 adult and 173 pediatric cases consisting of 197 astrocytic and 197 non-astrocytic tumors) to determine the frequency of polymorphisms in the GSTM1, GSTT1, and GSTP1 genes compared to a healthy control population. Our data shows that the frequency of GST polymorphisms varies not only between adult and pediatric patients with brain tumors and healthy controls, but also between different histological subtypes of brain tumors occurring in pediatric patients. We found (i) a statistically significant increase in the frequency of the functional GSTM1 allele in high-grade pediatric astrocytomas (p < 0.002), (ii) a significant increase in the frequency of the rare GSTP1 variant Val114/Val114 in pediatric astrocytomas (p < 0.002), and (iii) a significant increase in the frequency of the rare GSTP1 Val114/Val114 genotype among pediatric tumors showing microsatellite instability (MSI) due to defects in mismatch repair (MMR) proteins (p = 0.003). Our results suggest that GSTM1 and GSTP1 polymorphisms may play a role in brain tumor susceptibility by histological subtype, particularly high-grade pediatric astrocytomas. Moreover, the presence of the genetic modifier GSTP1 Val114/Val14 genotype may begin to define a high-risk genotype for cancer susceptibility in the pediatric population.