The cucumber (Cucumis sativus L.) is an important crop worldwide. In the present study, the molecular genetic diversity of 131 Spanish accessions was analyzed using 23 simple sequence repeat (SSRs). Eighteen of these SSRs were polymorphic; the mean number of alleles, mean observed heterozygosity and mean polymorphic information content were 3.2, 0.065 and 0.229, respectively. Seven SSRs showed a polymorphic information content (PIC) ranging from 0.31 to 0.44, therefore they were reasonably informative. Around 60% of the alleles showed a frequency higher than 0.05, and only one allele in the SSR31399 showed a frequency lower than 0.01. In addition, three accession-specific alleles were found. A high proportion of variation among accessions was obtained. In no case all plants of any accession showed the same genotype and only 18 of 131 Spanish accessions had at least two plants with the same genotype. A cluster analysis did not show any relation with morphological types or geographical area. Therefore, these results demonstrated that molecular diversity of the cucumber did not reflect its phenotypic variability. Finally, this study provided information for the rationalization of the cucumber collection of the COMAV. Morphological traits, origin and molecular data were taken into account to select 47 accessions, six belonging to ‘French’ type, 15 to ‘Long’ type, and 24 to ‘Short’ type. Phenotypic and molecular variability contained in the complete collection was conserved in the selected accessions.