The present study was conducted in the Tissue Culture Lab., Department of Vegetable Crops, Faculty of Agriculture, CairoUniversity, Giza, Egypt, during the period from 2005 to 2008. The objective of this work was to produce tetrahaploid plants from strawberry (Fragaria × ananassa Duch.) by culturing unpollinated ovaries of two different genotypes, viz., Camarosa and Sweet Charlie in vitro. Floral buds, 2-4 days before anthesis, were pre-treated at 4˚C for different periods and cultured on different media composition. Germinated ovules were performed and multiplication was induced to produce new shoots. Shoots were rooted and produced whole plantlets for adaptation. Results showed that cold pre-treatment was not efficient for producing the best ovule regeneration and production of tetrahaploid plants. Both cultivars gave nearly similar results under the optimal conditions for ovule regeneration. Determination of ploidy level was carried out and examinations showed that plantlets produced from the ovule culture were tetrahaploid. These results represent a modern tool for breeders to produce tetrahaploid strawberry plants to develop new cultivars in a short period (few months), or using the protocol of ovule regeneration in genetic engineering experiments by transferring new genes to develop better yield or quality of strawberry cultivars.