Introduction The purpose of this study was to compare digital periapical and cone beam computed tomography (CBCT) images to determine the number of canals in the mesiobuccal root (MB) of maxillary molars and to compare these counts to micro CT (μCT), which was also used to determine canal configuration. Methods Digital periapical (RVG 6100), CBCT (9000 3D) and μCT images (the reference standard) were obtained of 18 hemi-maxillas. With periapical and CBCT images, 2 endodontists independently counted the number of canals in each molar and repeated counts 2 weeks later. Teeth were extracted, scanned with μCT, and 2 additional endodontists, by consensus, determined the number and configuration of canals. The Friedman test was used to test for differences. Results In mesiobuccal roots, 2 canals were present in 100% (13/13) of maxillary first and 57% (8/14) second molars, and 69% (9/13) and 100% (8/8) of these exited as two or more foramina. There was no difference in canal counts for original and repeat reads by the two observers with periapicals (P = 0.06) and with CBCT (P = 0.88) and no difference when CBCT counts were compared with μCT counts (P = 0.52); however, when periapical counts were compared with μCT counts there was a significant difference (P = 0.04). Conclusions For cadaver maxillary molars, μCT canal counts were significantly different from digital periapical radiograph counts but not different from Carestream 9000 3D CBCT counts.