This study investigated the importance of comprehensive genetic diagnosis in pediatric B-cell acute lymphoblastic leukemia (B-ALL). We analyzed 175 B-ALL employing karyotyping, FISH, MLPA, targeted next-generation sequencing (t-NGS), and Optical Genome Mapping (OGM). This approach achieved an 83% classification rate, identifying 17 distinct genetic subtypes. Specifically, within B-other subtype, seven different subgroups were identified (ZNF384, IGH, DUX4, NUTM1 rearrangements, PAX5 alterations, PAX5 P80R, and IKZF1 N159Y). Secondary genetic alterations were observed, with copy number alterations (CNA) present in 60% of cases and mutations detected in 70.6%. While these alterations exhibited specific associations with certain genetic subtypes, CNAs did not appear to significantly impact the prognosis within these genetic groups. HeH, ETV6::RUNX1, ZNF384r, and PAX5 P80R exhibited excellent outcomes, contrasting with the poor prognoses observed in KMT2Ar, hypodiploidy, and CRLF2r (5-year overall OS were 50%, 50%, and 52%, respectively). These findings underscore the value of integrated genetic diagnostics for accurate subtyping, risk stratification, and guiding personalized treatment in pediatric B-ALL. Therefore, optimizing diagnostic workflows for routine clinical practice is crucial. Our study confirms the utility of conventional techniques (karyotyping and FISH), combined with t-NGS and OGM, for comprehensive genetic diagnosis.