Spinach (Spinacia oleracea L.) belongs to the Amaranthaceae family and is a popular and nutritious vegetable. It is believed that this green leafy plant originated from Persia which is now modern Iran and neighboring countries. In this study we screened 176 spinach world collection germplasm accessions with 15 known SSR markers. The markers produced 58 bands with 57 identified as polymorphic. PIC values of the markers ranged between 0.01 and 0.44. Average PIC value was 0.28. Based on allele analysis with Darwin5 and STRUCTURE tools, 176 individual were clustered into three groups. The first cluster mostly consisted of accessions from Europe and USA and contained 69 samples. The second cluster mostly consisted of lines from Asia and neighboring countries and contained 89 samples. The third cluster did not represent any characteristic according to geographic region, thus it was called an intermixed cluster which contained 18 samples. The maximum genetic dissimilarity of spinach accessions was 0.551 and minimum was 0.019. Average genetic dissimilarity was 0.217. Moreover we sequenced S. oleracea L. cv. Universal nuclear genome via IIIumina MiSeq technology and genome assembly was performed to develop new spinach–specific SSR markers. As a result, 3853 SSRs were identified in the assembled genome and we successfully designed 3275 primer pairs for these identified SSR motifs. These newly developed SSR markers will be helpful to scientists who are interested in spinach genome diversity and breeding.