l-asparaginase is an effective antineoplastic agent used in chemotherapy of acute lymphoblastic leukemia. The drug effect may be compromised by an elicited immune response, resulting in the production of anti-asparaginase antibodies causing an anaphylactic reaction or silent inactivation of the enzyme. To elucidate possible genetic predisposition for inter-individual differences in asparaginase hypersensitivity, we studied single nucleotide polymorphisms (SNPs) in the GRIA1 gene in 146 pediatric patients treated with l-asparaginase. Allergic reaction to l-asparaginase occurred in 49.3% of patients. We observed a statistically significant association between SNPs in the GRIA1 gene and the occurrence of asparaginase allergy: rs4958351 with p = 0.003, rs4958676 with p = 0.005, rs6889909 with p = 0.005, rs6890057 with p = 0.005 and rs10070447 with p = 0.006. We found a statistically significant correlation between asparaginase allergy and event-free survival (p-value 0.005).