[Objective] Fowlpox virus (FPV) is a member of Avipoxvirus. FPV is widely used as a living virus vector in poultry and mammals because of its large genome and containing large numbers of replication non-essential regions. The selection of recombination sites is a prerequisite to construct fowlpox virus vectors. The insertion of foreign genes that do not affect viral replication is a prerequisite for selection of the insertion sites. Therefore, identification of replication non-essential regions for foreign gene insertion would provide additional options to construct recombinant viruses. This study aimed to identify the necessity of thymidine kinase (TK) gene in the replication of FPV NX10. [Methods] The recombinant virus rFPV-ΔTK-EGFP was acquired by homologous recombination using FPV NX10 strain as parental virus and enhanced green fluorescent protein (EGFP) as selection marker. The bromodeoxyuridine (BUdR) was added to the culture of CEF cells to identify the role of TK in FPV replication. [Results] The transfer vector pUC19-TK AB-EGFP was constructed. Although the green fluorescent lesions increased in the cloning and purification of recombinant virus, non-fluorescent lesions can still be observed at the edge of the fluorescent plaque. Western blot analysis of the randomly selected plaque clones after 9 to 15 rounds showed that the EGFP gene inserted in the recombinant virus could be expressed correctly, but PCR results showed that the wild-type viruses accompanied with the recombinant virus. After adding BUdR to the cell culture medium, the recombinant virus could not continue to replicate. [Conclusion] The TK gene of FPV NX10 strain is partly essential for replication.