Acute lymphoblastic leukemia (ALL) can be classified into different subgroups based on recurrent genetic alterations. Here, targeted RNA-sequencing was used to identify the novel subgroups of ALL in 144 B-other and 40 'classical' ALL samples. The 'classical' TCF3-PBX1, ETV6-RUNX1, KMT2A-rearranged, BCR-ABL1, and novel P2RY8-CRLF2, ABL-, JAK2-, ZNF384-, MEF2D-, and NUTM1-fusions were easily identified by fusion transcript analysis. IGH-CRLF2 and IGH-EPOR were found by abnormally high levels of expression of CRLF2 or EPOR. DUX4-rearranged were identified by the unusual expression of DUX4 genes and an alternative exon of ERG, or by clustering analysis of gene expression. PAX5-driven ALL, including fusions, intragenic amplifications and mutations were identified by SNV analysis and manual inspection using the IGV software. Exon junction analysis allowed detection of some intragenic ERG and IKZF1 deletions. CRLF2-high associated with initial white blood cell (WBC) ≥50,000/L and GATA3 risk alleles (rs3781093 and rs3824662), while ABL/JAK2/EPOR-fusions associated with high WBC, NCI high risk and IKZF1del. ZNF384-fusions associated with CALLA-negativity and NUTM1-fusions with infants. In conclusion, Targeted RNA-sequencing further classified 96/144 (66.7%) B-other cases. All novel ALL subgroups, except for iAMP21, hyper- and hypodiploid cases were identified. Curiously, we observed higher frequencies of girls within B-'rest' ALLs and boys in PAX5-driven cases.-