Summary Reasons for performing study The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objectives The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1–2 × 10 9 cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37°C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel. Results Average cell viability upon thawing was 98.7 ± 0.6%. In 6 out of 7 samples, adherent spindle‐shaped cell colonies were observed within 9.0 ± 2.6 days and attained 80% confluency at 12.3 ± 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD 29, CD 44 and CD 90 positive, and CD 45, CD 73, CD 79α, CD 105, MHC II and monocyte‐marker negative. Conclusions and potential relevance Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time‐ as well as cost‐efficient approach in equine regenerative medicine.