Objective: To evaluate a template-based matching algorithm on single-energy (SE) and dual-energy (DE) radiographs for markerless localization of lung tumours. Methods: A total of 74 images from 17 patients with Stages IA-IV lung cancer were considered. At the time of radiotherapy treatment, gated end-expiration SE radiographs were obtained at 60 and 120 kVp at different gantry angles (33°anterior and 41°oblique), from which soft-tissue-enhanced DE images were created. A template-based matching algorithm was used to localize individual tumours on both SE and DE radiographs. Tumour centroid co-ordinates obtained from the template-matching software on both SE and DE images were compared with co-ordinates defined by physicians.Results: The template-based matching algorithm was able to successfully localize the gross tumor volume within 5 mm on 70% (52/74) of the SE images vs 91% (66/ 74) of the DE images (p , 0.01). The mean vector differences between the co-ordinates of the template matched by the algorithm and the co-ordinates of the physician-defined ground truth were 3.2 6 2.8 mm for SE images vs 2.3 6 1.7 mm for DE images (p 5 0.03). Conclusion: Template-based matching on DE images was more accurate and precise than using SE images. Advances in knowledge: This represents, to the authors' knowledge, the largest study evaluating template matching on clinical SE and DE images, considering not only anterior gantry angles but also oblique angles, suggesting a novel lung tumour matching technique using DE subtraction that is reliable, accurate and precise.