This experiment was performed on cucumber (Cucumis sativus L.) at Research Farm of the Faculty of Agriculture, Al-Azhar University (Assiut Branch), Assiut, Egypt (Longitude 031° 11' 09" E and latitude 27° 10' 39" N) in sandy clay loam soil. To evaluate nine F₁ hybrids of cucumber by using molecular analysis. Cucumber hybrids were planted in March 2019 and 2020 for the summer seasons 2019 and 2020. The experiment was laid out in Randomized Complete Block Design (RCBD) with three replications. Randomly amplified polymorphic DNA was used to assess the genetic diversity and relationship among 9 cucumber hybrids. Five RAPD primers generated a total number of 34 bands, out of them 3 bands were polymorphic with 8.82% polymorphism. The total number of bands generated by each primer ranged from 5 to 9 bands with an average of 6.8 band per primer. Additionally, only two primers gave polymorphism among cucumber samples i.e., OPA-03 and OPA-08. Some genetic diversity measures were estimated based on RAPD molecular data. In this regard, the polymorphism information content (PIC) ranged for 0.05 (OPA-03) to 0.16 (OPA-08) with an average of 0.04 for all primers. The primer resolving power was 0.67 for OPA-03 and 1.11 for OPA-08, with an overall value of 1.78 for RAPD primers. In addition, diversity index was almost the same as PIC, with 0.04 (OPA-03) and 0.16 (OPA-08) and an average of 0.04 for all RAPD primers.