Studies have shown the power of transcriptome sequencing [RNA sequencing (RNA-Seq)] in identifying known and novel oncogenic drivers and molecular subtypes of B-cell acute lymphoblastic leukemia (B-ALL). We hypothesized that our clinically validated RNA-Seq assay, coupled with a custom analysis pipeline, could be applied to comprehensive B-ALL classification. RNA-Seq was performed on 76 retrospective B-ALL cases, 28 with known and 48 with undetermined subtype, following clinical karyotype analysis, fluorescence in situ hybridization, chromosomal microarray, and next-generation sequencing panel testing (OncoKids). Subtypes were accurately identified in all 28 known cases, and they were determined in 38 of 48 unknown cases (79%). The subtypes of the unknown cases included the following: PAX5alt (n = 12), DUX4 rearranged (n = 6), Philadelphia chromosome-like (n = 5), low hyperdiploid (n = 4), ETV6:RUNX1-like (n = 3), MEF2Dr (n = 2), PAX5 P80R (n = 2), ZEB2/CEBP (n = 1), NUTM1r (n = 1), ZNF384r (n = 1), and TCF3:PBX1 (n = 1). In 15 of 38 cases (39%), classification based on expression profile was corroborated by detection of subtype-defining oncogenic drivers missed by clinical testing. Among the 10 remaining unclassified cases by RNA-Seq, 4 had suboptimal samples (2: 70% post-transplant donor chimerism). RNA-Seq analysis also detected large copy number abnormalities, oncogenic hot-spot sequence variants, and intragenic IKZF1 deletions. Our pilot study confirms the feasibility of implementing an RNA-Seq workflow for clinical diagnosis of molecular subtypes in pediatric B-ALL, reinforcing that RNA-Seq represents a promising global genomic assay for this heterogeneous leukemia.