The objective of the present study was to conduct a corresponding histological analysis of 162 macroscopically assessed keel bones (1: severe, 2: moderate, 3: slight, 4: no deformity). Four layer lines were used and hens were kept in furnished cages, small group systems (both allowing more activities due to the provision of perches) and an aviary system, which fully conformed to the EU standards. Investigations were carried out in the 3rd, 6th, 9th and 12th laying month of two experimental trials. In 97.9% of grade 4 keel bones, no histological deviations were found, whereas in keel bones manifesting deformities of grade 1 and 2, the predominant histological observation was the incidence of fracture callus material (FCM) and new bone in the form of woven bone. FCM was also detected in 50.9% of grade 3 keel bones, whereas in 40.7%, only s-shaped deviations of keel bones were found, which were related to extended pressure loading while perching activities rather than short-duration trauma. Histological analysis showed that keel bones of grade 1 and 2 were mainly attributed to traumatic origin and therefore associated with pain experience in layers. Grade 3 keel bones manifested either FCM as a result of trauma or adaptational deformities without any evidence of a preceding fracture in response to mechanical pressure loading and were most likely not associated with pain. Therefore, histological analysis was found to be a mandatory tool when evaluating grade 3 keel bones with respect to layers'welfare. Furthermore, this analysis corroborates the findings that in aviary systems deformities of keel bones are predominantly caused by painful fractures.