This study investigated the association between genetic variants and the occurrence of oral mucositis (OM) in pediatric patients with acute lymphoblastic leukemia (ALL) and lymphoma undergoing chemotherapy (CT). A retrospective observational longitudinal study was conducted, with daily assessment of OM using the World Health Organization scale and blood samples collected for DNA extraction. Genetic variants were analyzed through next-generation sequencing, using a gene panel focused on 67 coding regions within 20 genes. Sixty-four pediatric patients were evaluated during 392 cycles of CT, with the most commonly used protocols being DOXO (34.2%), MTX (27.8%), and CTX (17.3%). Approximately 65.8% of patients developed some degree of mucositis, with 34.7% showing ulcerative OM (Grades 2 or 3) and 9.2% presenting severe OM (Grade 3). Associations between genetic variants and OM were identified in MTX cycles (ABCC2, ABCC4, and GSTM1), CTX cycles (ABCC6, HSP90AA1, and ABCC1), and DOXO cycles (ABCC1, CYP2A7, and MTHFR). This study suggests potential future treatment strategies with the possibility of increasing efficacy, reducing toxicity, and improving survival rates in pediatric oncology patients.