Spinach (Spinacia oleracea L.) is an economically important crop that is cultivated and consumed worldwide. Spinach is interfertile with the wild species S. tetrandra Steven ex M. Bieb. and S. turkestanica Iljin that therefore are presumed to include the most likely crop ancestor. Here we studied variation in 60 Single Nucleotide Polymorphisms (SNP) previously identified in S. oleracea to address the issue of crop ancestry and domestication region. For this purpose we investigated 95 accessions, including 54 spinach landraces from a wide geographic area in Europe and Asia and 16 S. tetrandra and 25 S. turkestanica populations of which the majority had only recently become available. Compared to S. tetrandra substantially higher levels of amplification success and higher levels of variation were detected for S. turkestanica, indicating that S. oleracea is genetically closer to S. turkestanica than to S. tetrandra. Our phylogenetic and population structure analysis supported the conclusion that S. turkestanica is the most likely ancestor of cultivated spinach. In addition, these analyses revealed a group of S. oleracea landraces from Eastern and Southern Asia with a strong genetic resemblance to S. turkestanica. This group includes landraces from Afghanistan and Pakistan, which are part of the native distribution range of S. turkestanica. The domestication of spinach may therefore have occurred more eastwards than generally assumed. Furthermore, our study provides support for the hypothesis that after domestication, spinach was introduced into China via Nepal. Additional collecting of spinach landraces is recommended in order to allow the more precise reconstruction of the crop migration routes.