B-cell acute lymphoblastic leukemia (B-ALL) is characterized by the malignant burgeoning of abnormal B-cell lymphoblasts. In recent years, the use of CART therapy which targets CD19 antigen present on the surface of B-cells, has gained significant attention as a treatment option against aggressive and refractory forms of B-ALL. However, the loss of CD19 antigen on B-cell surface due to aberrant splicing under therapy pressure has been suggested as one of the main factors for the emerging CART therapy resistance. Herein, using RT-PCR based splice assays we examined CD19 splicing patterns in 43 primary pediatric B-ALL patient samples spread across various subtypes. We observed that CD19 isoform lacking exon 5-6 exists in ~ 55% of pediatric patients at the initial diagnosis stage itself. Using in-silico analysis, we identified RNA binding proteins, RC3H1 and MBNL1, as potential regulators of exon 5-6 skipping. Furthermore, qRT-PCR analysis in patient samples revealed that RC3H1 and MBNL1 are significantly upregulated in samples exhibiting exon 5-6 skipping. Taken together, we for the first time report the existence of aberrantly spliced CD19 isoform lacking exon 5-6 in primary pediatric patients, and this occurrence could potentially result from RC3H1 and MBNL1 dysregulation.