The potential use of neural stem cells (NSCs) in basic research, drug testing, and for the development of therapeutic strategies is dependent on their large scale in vitro amplification which, however, introduces considerable risks of genetic instability and transformation. NSCs have been derived from different sources, but the occurrence of chromosomal instability has been monitored only to a limited extent in relationship to the source of derivation, growth procedure, long-term culture, and genetic manipulation. Here we have systematically investigated the effect of these parameters on the chromosomal stability of pure populations of mouse NSCs obtained after neuralization from embryonic stem cells (ESCs) or directly from fetal or adult mouse brain. We found that the procedure of NSCs establishment is not accompanied by genetic instability and chromosomal aberration. On the contrary, we observed that a composite karyotype appears in NSCs above extensive passaging. This phenomenon is more evident in ESC- and adult sub-ventricular zone-derived NSCs and further deteriorates after genetic engineering of the cells. Fetal-derived NSCs showed the greatest euploidy state with negligible clonal structural aberrations, but persistent clonal numerical abnormalities. It was previously published that long-term passaged ESC- and adult sub-ventricular zone-derived NSCs did not show any defects in the cells' proliferative and differentiative capacity nor induced in vivo tumour formation, although we here report on the chromosomal abnormalities of these cells. Although chromosomal aberrations are known to occur less frequently in human cells, studies performed on murine stem cells provide an important complement to understand the biological events occurring in human lines.