Acute lymphoblastic leukemia (ALL) is the most common childhood cancer worldwide, and exhibits high molecular heterogeneity. Molecular subtypes are characterized by specific chromosomal and molecular alterations, which are critical for guiding risk-adapted therapies. However, the increasing number of recognized prognostic molecular subtypes demands large resources, which are often limited in low/middle-income countries; thereby restricting the molecular characterization. This study aimed to perform an integrated molecular characterization of childhood B-ALL in Argentine patients. We performed RNA-seq on diagnostic bone marrow aspirates from Argentine patients enrolled in the ALLIC-GATLA-2010 protocol. We used different bioinformatic tools to identify and validate single nucleotide variants, fusion transcripts, gene expression profiles and molecular subtypes. We successfully determined transcriptome-based molecular subtype in 93.7% of patients; with high concordance to conventional karyotyping and RT-PCR (17/18 patients with available molecular data). Analysis of chimeric transcripts revealed 82 fusions, both intra– and inter-chromosomal, suggesting that leukemic cells may undergo chromosomal instability. Two of these fusions were novel: SCAF8::FER1L4 and DBF4B::EFTUD2. We also identified 21 different SNVs/InDels in 16 genes, including three novel variants (DUX4 p.I65N, CREBBP p.G1542V, and CSF3R p.G147R) and predicted to alter protein function. Overall, we observed that all patients who relapsed carried high-risk genetic alterations at diagnosis. Whole-transcriptome analysis of leukemic bone marrow enabled molecular subtyping and the identification of both known and novel molecular alterations associated with prognosis.