Introduction/Objective: PEG-Asparaginase (PEG-ASNase) is critical in treating pediatric acute lymphoblastic leukemia (ALL). However, hypersensitivity reactions and inactivations associated with ASNase are major patient challenges. The detection of single nucleotide polymorphisms (SNPs), which recognize, in advance, patients who will develop hypersensitivity to ASNase, would help to optimize treatment. To describe pharmacogenetic variations influencing hypersensitivity to PEG-ASNase and their correlation with our patient population. Material and methods: We conducted a prospective multicenter study involving ALL patients under 18 years old receiving PEG-ASNase. Genotyping was carried out for 6 SNPs, which are more frequently related to hypersensitivity in other populations, including GRIA1 rs4958351, NFACT2 rs6021191, MYBBP1A rs3809849, CNOT3 rs73062673, GR1A1 rs6890057 and HLA-DQB1 rs1694129. The tests were performed using custom TaqMan® SNP genotyping assays. Results: A total of 305 patients were included, with 10.8% experiencing clinical allergic reactions and (14.1%) patients with inactivation (ASNase activity 0.01). Discussion: Our analysis characterized the pharmacogenetic polymorphisms in our population, reflecting diversity similar to the general population. Despite the lack of correlation between the studied SNPs and allergic reactions observed in our sample, we encourage further exploration to identify additional SNPs associated with hypersensitivity. Conclusion: These findings lay the groundwork for future research to uncover new genetic predictors of hypersensitivity, which could contribute to the development of personalized treatment approaches and the prevention of allergic reactions.