Abstract: Thymidylate kinase catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis, and it is crucial for DNA replication and life survival. In previous study, the tmk-a-1, tmk-a-2 and tmk-b genes of Paulownia witches'-broom Pingshan strain (PaWBPS) were obtained. On the basis of previous study, the amino acid sequence alignment and similarity analysis were conducted among PaWBPS TMK-a-1, PaWBPS TMK-a-2, PaWBPS TMK-b, onion yellow wild-type line (OY-W) TMK-a, OY-W TMK-b, white blue dwarf (WBD) TMK-a and WBD TMK-b. The amino acid sequence similarity value was 96.65% between PaWBPS TMK-b and WBD TMK-2, 99.03% between PaWBPS TMK-b and OY-W TMK-b, 90.57% between PaWBPS TMK-a-1 and PaWBPS TMK-a-2, ranged from 87.32% to 99.26% among PaWBPS TMK-a-1, PaWBPS TMK-a-2, WBD TMK-1 and OY-W TMK-a, and from 22.22% to 25.95% among TMK-a and TMK-b of PaWBPS, OY-W and WBD phytoplasma. The pET28a system was used to generate a polyhistidine (polyHis)-tagged TMK fusion protein. The polyHis-tagged TMKs were expressed in Escherichia coli BL21 (DE3) cell and purified under native conditions by Ni-NTA chelating column. Then, the TMK activity was measured using enzyme-coupled assay. Results suggested that the TMK-b had thymidylate kinase activity (85.96±0.74 U·mg-1) and the TMK-a-1 and TMK-a-2 had hardly any activity.