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Genetic maps with a high density of molecular markers are essential for plant breeding. SSR (Simple Sequence Repeat) is a molecular marker for creating genetic maps and distinguishing heterozygous and homozygous plants. The main aim of this study was to construct SSRs associated with key traits in cucumber breedings and to determine the optimum conditions for PCR reactions to amplify cucumber DNA utilizing these SSRs. An in silico survey was performed to mine the SSR data set. Optimization of PCR reaction by running temperature gradients for all the SSR primers. We identified 52 SSRs that are associated with important traits in cucumber breeding. Also, the optimal annealing temperatures for PCR of 46 SSRs were determined. These are important data for the next screening steps for segregating plants for further cucumber breeding.