The evaluation of genetic diversity is crucial for breeders to develop strategies and improve the resilience, quality, and adaptability of the date palm. In this study, the genetic diversity of three date palm varieties was performed using ISSR-PCR molecular markers to determine its relationship with in vitro propagation response of these varieties. The molecular profiling was performed using ISSR-PCR. A total of 49 loci were produced by the PCR reactions, 38 of which were polymorphic while 11 were monomorphic. The level of polymorphism revealed by ISSR-PCR varied from 33.33% to 100%. The three date palm varieties were grouped into two clusters based on the results of cluster analyses that used morphological data and molecular profiles. Cluster I comprised the ‘Barhy’ variety and Cluster II included ‘Magdoul’ and ‘Amri’ varieties. The clustering analyses revealed the independence of the ‘Barhy’ variety in its characteristics from the other varieties based on either morphological or molecular data. The results of in vitro propagation showed that the ‘Amri’ variety exhibited the highest callus induction frequency (86.28%), callus weight (2.33 g), number of somatic embryos (9.32), number of shoots (14.62), number of roots (4.11), root length (4.63 cm), shoot length (13.61 cm) followed by ‘Magdoul’ and ‘Barhy’ varieties. The ‘Amri’ variety had the shortest callus induction period, at 23.26 days while the ‘Barhy’ variety exhibited the longest period of callus induction (28.55). It was deduced from the study that the ISSR marker reproduced trustworthy patterns of bands to determine the genetic diversity among different date palm varieties that are considered the cornerstone for the genetic improvement of date palms. The understanding of the relationship between genetic diversity and in vitro propagation response of date palm is essential for ensuring the long-term sustainability of its crop. This will facilitate better conservation and development of new date palm varieties that fulfil the needs of farmers and consumers.