Spinach (Spinacia oleracea L.) is a highly nutritious leafy vegetable and an economically important food crop. The wild species S. turkestanica Iljin and S. tetrandra Steven ex M. Bieb. are inter-fertile with cultivated spinach and constitute important sources of novel characters to improve spinach varieties, such as for their resistance to pests and diseases. Despite their relevance in plant breeding, S. turkestanica and S. tetrandra are poorly represented in genetic resources collections. Among the reasons for these collection gaps are the difficulties in propagating these species ex situ. Here we report on the results of collecting expeditions for S. turkestanica in Central Asia and for S. tetrandra in the Trans-Caucasus, which were organized by the Dutch gene bank in collaboration with several breeding companies. Furthermore, we also present efficient protocols for the ex situ regeneration of these species. These protocols were used to successfully regenerate 66 S. turkestanica and 36 S. tetrandra samples from the collecting expeditions. These new accessions fill up important collection gaps in ex situ conserved genetic resources of spinach and can be used for exploitation in crop improvement.