Current procedures to establish vertebral column regionalization (e.g., histology) in fish are time consuming and difficult to apply. The aim of this study was to develop a more rapid and accurate radiology-based method for Atlantic salmon (Salmo salar). A detailed analysis of 90 animals (4 kg) led to the establishment of region-specific radiographic hallmarks. To elucidate its transferability to other salmonid species, radiography was carried out in brown trout (Salmo trutta), Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus mykiss), pink salmon (Oncorhynchus gorbuscha), and Chinook salmon (Oncorhynchus tshawytscha). This method was also evaluated for whole ungutted fish. The vertebral column of Atlantic salmon can be subdivided into five regions (R1-R5) based on anatomy: postcranial (R1, V1, and V2), abdominal (R2, V3-V26), transitional (R3, V27-V36), caudal (R4, V37-V53), and ural (R5, V54-V59). The following specific radiographic hallmarks allow the identification of regions: (i) lack of ribs in R1, (ii) modified parapophysis of the first vertebra of R3, (iii) prominent hemal spine of the first vertebra of R4, and (iv) the separated hemal spine of the most cranial pre-ural vertebra of R5. These hallmarks were all transferable to the other salmonid species assessed. The results include a further description of various region-specific characteristics in Atlantic salmon. The method was found applicable for sedated/whole ungutted fish, verifying it as quick and easy compared to other regionalization methods. The regions defined by radiology in this study agree with the vertebral column regions recently defined for Chinook salmon (O. tshawytscha). Thus, and considering the results of this study on various salmonid species, the currently developed regionalization protocol can be generally used for salmonids.