Regenerative medicine and cell therapy are emerging clinical disciplines in the field of stem cell biology. The most important sources for cell transplantation are human embryonic and adult stem cells. The future use of these human stem cell lines in humans requires a guarantee of exhaustive control with respect to quality control, safety and traceability. Genetic instability and chromosomal abnormalities represent a potential weakness in basic studies and future therapeutic applications based on these stem cell lines, and may explain, at least in part, their usual tumourigenic properties. So, the introduction of the cytogenetic programme in the determination of the chromosomal stability is a key point in the establishment of the stem cell lines. The aim of this review is to provide readers with an up-to-date overview of all the cytogenetic techniques, both conventional methods and molecular fluorescence methods, to be used in a stem cell bank or other stem cell research centres. Thus, it is crucial to optimize and validate their use in the determination of the chromosomal stability of these stem cell lines, and assess the advantages and limitations of these cutting-edge cytogenetic technologies.