The reproducibility of immobilization is critical in small animal radiotherapy, especially for the head region. We developed a reusable immobilization method using a thermoplastic mask molded with cylinders that can be universally applied, eliminating the need for individual patient-specific masks. We aimed to compare the effectiveness of this method with two conventional techniques, namely immobilization using a cylinder and patient-specific thermoplastic mask immobilization. Five beagle dogs underwent repeated CT scans for each method, and the positional errors of the anatomical landmarks were measured. In addition to the maxilla, we examined the mandible and lymph nodes in the head and neck region, which have rarely been considered in previous studies. The results demonstrated that the accuracy of the new universal immobilization method was comparable with that of the conventional methods, with no significant differences in the positioning of the bony landmarks. Universal immobilization outperformed cylindrical immobilization for the lymph nodes. However, large errors were observed in the positions of the lymph nodes across all tested head immobilization methods. The universal method required the shortest setup time, emphasizing its efficiency. It also eliminated the need for patient-specific individual masks, making it simple and sustainable. Despite certain limitations, such as the exclusive use of beagle dogs and the single-day anesthesia protocol, the study concluded that the cylindrical thermoplastic mask is a promising method that offers precision and efficiency. This study represents a model investigation rather than a validated clinical approach. Further proof-of-principle investigations are required to validate the clinical utility of this approach.