Core activities of genebank operations include the preservation of germplasm identity and maintenance of genetic integrity. Some organisms such as banana are maintained by tissue culture that can foster accumulation of somatic mutations and loss of genetic integrity. Such changes can be reflected in their genome structure and thus be revealed by sequencing methods. Here, we propose a protocol for the detection of large chromosomal gains and/or losses that was applied to in vitro banana accessions with different levels of ploidy. Mixoploidy was detected in triploid (3x) accessions with chromosomal regions being diploid (2x) and tetraploid (4x) and in diploid accessions (2x) where large deletions resulted in partial haploidy (1x). Such abnormal molecular karyotypes can potentially explain phenotypic aberrations observed in off type material. With the affordable cost of Next Generation Sequencing (NGS) technologies and the release of the presented bioinformatic pipeline, we aim to promote the application of this methodology as a routine operation for genebank management as an important step to monitor the genetic integrity of distributed material. Moreover, genebank users can be also empowered to apply the methodology and check the molecular karyotype of the ordered material.