Background: Limited information comparing digital subtraction radiographic assessment with conventional radiographic interpretation is available from longitudinal clinical trials. The aim of this study was to evaluate the ability to detect periodontal bone changes during the long-term maintenance of Class II furcation defects by conventional radiographic interpretation compared to interpretation of digital subtraction images. Methods: Standardised radiographs of 18 Class II furcation defects in mandibular molars were taken at baseline and at 6, 12, 18, and 24 months after non-resective periodontal surgery. Conventional radiographic and digital subtraction interpretations were performed masked, respectively. by two and three experienced examiners, according to the following categories: bone gain; bone loss; unchanged appearance; and impossible to visualize. Percent concordance and the kappa statistic value (kappa) were computed. Results: Conventional radiographic and digital subtraction interpretation images resulted in 72 decisions for each examiner. The visual interpretation of digital subtraction images by two examiners revealed the same results. The interpretation of conventional radiographic images showed a low concordance between examiners (kappa <0.40) at all examinations. The concordance between subtraction radiography and conventional radiographic interpretation was also low for all examiners (kappa <0.36) at all examinations. Using subtraction radiography as a reference, bone changed and bone unchanged were diagnosed correctly in 47.2% of cases by examiner A, in 43.1% by examiner B, and in 38.9% by examiner C. Conclusion: It can be concluded that conventional radiographic interpretation is a more subjective and inaccurate method of detecting periodontal bone changes in Class II furcation defects in mandibular molars when compared with subtraction radiography.75811451149