Development of safe and effective radio frequency (RF) vessel sealing devices requires the complete characterization of thermal damage that can occur adjacent to areas of energy application. Hematoxylin and eosin (H&E) staining is an efficacious and extensively used histological test method for assessing this lateral thermal damage through characterization of changes in cell and tissue morphology. The purpose of this investigation was to compare thermal spread measurements obtained using H&E staining to measurements made using picrosirius red (PSR) staining, an alternative histological test method that exploits the birefringence of healthy vascular collagen to facilitate the identification of thermal damage associated with use of vessel sealing devices. A 5 mm RF vessel sealing device was used to seal arteries in vivo in various porcine tissues. Subsequently, sealed vessel segments were excised and histologically processed. Paired histological sections were mounted on slides; one section was stained with PSR and one section was stained with H&E. A blinded board-certified veterinary pathologist completed thermal spread measurements on all PSR- and H&E-stained sections. Bland–Altman comparison plots were then used to estimate any bias between the two measurement methods, as well as quantify the range of differences between PSR and H&E thermal spread measurements. The magnitude of the mean difference between measurements was determined to be less than 0.10 mm and, with 90 % confidence, 95 % of the differences between measurements were within 1.0 mm. These results indicate that PSR-staining is an appropriate alternative to H&E-staining for the measurement of lateral thermal spread associated with an RF vessel sealing device.