Abstract: Regeneration efficiency and genetic clonality are two major aspect of successful long-term in vitro culture to be studied extensively. The regeneration efficiency of 25 months sustained in vitro culture of Allium ampeloprassum L. was maintained through multiple shoot proliferation and root growth. No morphological variation was detected between the plantlets regenerated from initial and long-term shoot tip culture or with their mother, meaning a high likelihood of their clonal fidelity. Detection of genetic integrity for in vitro regenerated clones was carried out using 10 ISSR primers among which 4 primers produced a total number of 336 distinct and scorable bands with an average of 7 bands per primers where the other 6 primers were not reproducible. The ISSR primers base on AG motif and 3' anchoring produced more number of consistent bands. All monomorphic bands in the ISSR assay both for primary culture as well long term culture in vitro ascertained to a great extent their genetic integrity through their partial genetic coverage.