Background: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer worldwide. Developed countries have a 90% 5-year overall survival rate with proper treatment, while LMICs have a poor rate of around 30-50%. The research aims to identify mutations in frequently mutated genes’ hotspot regions to design appropriate treatment plans based on patients’ somatic makeup. Methods: Sanger sequencing was conducted on TP53, PAX5, and JAK2 gene hotspot regions in 60 Patients with ALL diagnosed with acute lymphoblastic leukemia, categorized into B-ALL and T-ALL subtypes. Results: The exon mutation rate was 8.33%. The mutation frequency for PAX5 was 5%, while for TP53, it was 3.33%. New mutations found in TP53 and PAX5 genes intron region. None of these mutations was found significant to have a poor prognosis either on the whole cohort or chemotherapy recipient patients. Among the mutated samples, Chr17:7674089 (A→C) and Chr17:7674109 (G→A) were found to have a worse prognosis in patients diagnosed with T-ALL. Chemotherapy treatment response is significant with p = 0.011, and there was a linkage between chemotherapy response and the overall mutation in chemotherapy patients (p=0.0013). The TP53 mutation in chemotherapy patients is related to poor survival (p=0.001) rather than the PAX5 mutation (p=0.087). Conclusion: TP53 gene mutation is associated with poor chemotherapy response, and subtypes specific study is required for the precise treatment plan for Bangladeshi pediatric patients with ALL.