Background: The clonal basis of relapse in acute lymphoblastic leukemia (ALL) is complex and not fully understood. Methods: Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex-ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis-relapse samples from 13 B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. Findings: The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (median, 6 alterations per sample) relative to that at diagnosis (median, 47 alterations) (p = 0.019). The combination of MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. Notably, TP53 was the most frequently mutated gene at relapse (31%). Two TP53 mutations were detected only at relapse, whereas the three others showed an increase of their mutational burden at relapse. Interpretation: Clonal evolution patterns were heterogeneous, involving acquisition, loss and maintenance of lesions at relapse. Therefore, this study confirmed that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. The combination of the NGS, aCGH, and MLPA approaches enables better molecular characterization of the genetic profile in ALL patients during the evolution from diagnosis to relapse. Funding Statement: This work was supported in part by a grant from the Consejeria de Educacion, Junta de Castilla y Leon, Fondos FEDER (SA085U16, SA271P18), Proyectos de Investigacion de la Gerencia Regional de Sanidad, SACYL, (GRS 1847/A/18), Fundacion Castellano Leonesa de Hematologia y Hemoterapia (FUCALHH 2017), a grant to AM from the Junta Provincial de Salamanca of the Asociacion Espanola Contra el Cancer (AECC), a grant to MFC from the Universidad Pedagogica y Tecnologica de Colombia – Vicerrectoria de Investigacion y Extension (Grupo de Investigacion en Ciencias Biomedicas UPTC – GICBUPTC, Escuela de Ciencias Biologicas). M. Hernandez-Sanchez is supported by FEHH-Janssen (Sociedad Espanola de Hematologia y Hemoterrapia), and a grant to JR and JMR from Instituto Carlos III (PI14/01971). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the local ethical committee, the Comite Etico de Investigacion Clinica, at the Hospital Universitario de Salamanca. Written informed consent was obtained from each patient or their legal guardian before entering the study.