Aim: to study the canal anatomy and its variations of mesiobuccal root of maxillary first molar using visual, CT, CBCT, and staining and clearing. Materials and Methods: Three hundred extracted permanent maxillary first molar teeth were selected for this study. Mesio-buccal roots were resected 1 mm coronal to the trifurcation level. The orifices of the canals were located with a size 10 K-type file which was passively advanced into the canals until the tip of the instrument penetrated the apical foramen for visual examination. The number of orifices and apical foramina in each root were recorded. All roots were embedded in a sheet of pink wax with vertical orientation and arranged in their numbers to facilitate three dimensional CT and CBCT scan and the data were stored on a magnetic optical disc. Both, CT and CBCT images were assessed by a calibrated dental radiology specialist blinded to the order of roots using Vitrea 2 V 3.8 imaging software. India ink was injected into the orifices of the root canals with a plastic disposable endodontic irrigating syringe with a 27-G needle with suction tip which was placed at the root apex to draw the ink through the root canal system. All roots were decalcified with 5% hydrochloric acid for three days, dehydrated in ascending concentrations of ethanol (75%, 85%, 96% and 100%) for four hours each, and transparent specimens were obtained by immersing the dehydrated roots in methyl salicylate solution, in which the roots were stored until they were examined. The data regarding root canal morphology of each sample from all evaluation methods were tabulated and compared statistically. Results: There were significant differences between the four methods used for studying the root canal anatomy of mesiobuccal root of first maxillary molars as regarding the number of canals, number of orifices, number of apical foramina, presence of lateral accessory canals and intercanal communications. Roots with three canals and three orifices and opened apically with two foramina showed no significant differences between the four methods. Also, with Kappa test, there was good and fair agreement between CBCT & CT and CBCT & staining and clearing respectively. Conclusion: the most detailed information can be obtained in-vitro by staining and clearing and high resolution CT and CBCT methods which are commonly used as a diagnostic aid in clinical endodontics