Jaw bone density measurements are applicable in many clinical situations to assess bone tissue. To be able to implement research findings in clinical reality, tools must be simple and low cost. Intra-oral radiographs including a reference material perform well as a densitometric tool. However, the inclusion of a reference material, usually in the form of a metal wedge, is an additional burden for the dentist. The aim of this study was to evaluate whether a reference step wedge is required for accurate densitometric results. Dual energy X-ray absorptiometry measurements and densitometric measurements on intra-oral radiographs using a custom-made software were performed on bone samples from the premolar region of the mandible. Observer agreement of bone density expressed as grey value was high. The correlation between mandibular bone mineral density and the densitometric values on intra-oral radiographs was substantially higher when the aluminium step wedge was included. The Wilcoxon test revealed no significant difference between the density measurements using nine or three steps of the Al reference wedge. Density determination of grey value and mm Aleq thickness value both have good intra- and inter-observer agreement. However, jaw bone densitometry is far more accurate when including a reference wedge.