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In the present investigation, 13 genotypes of cucumber were screened for genetic diversity using eight ISSR primers. The six ISSR primers generated 52 alleles. A total of 52 loci were amplified that exhibited 92.30 per cent polymorphism. A maximum of 11 loci were detected by the primer UBC-855 and UBC-890. Unique bands were also observed by the primers namely UBC-808, UBC-840, and UBC-855. The similarity value ranged from 22 per cent to 80 per cent with the average value of 46 per cent. The genotype Pgyn-1 was found most diverse than others with 33 per cent similarity. The 13 genotypes were grouped into two major groups (A and B) based on ISSR markers. Group A consisted of the most diverse genotype, Pgyn-1. Group B contained the maximum number of genotypes and further divided in two major sub clusters IB and IIB. Sub cluster IB includes small sub cluster of seven genotypes in which Pgyn-4 was found separately whereas, sub cluster IIB contained PCUC-35 and Punjab Naveen as well as shared the cluster genetically while they differ a lot in respect to visual identification.