The purpose of this study was to evaluate the incidence and microscopic anatomy of the isthmus to provide more precise anatomical information about the mesiobuccal (MB) roots of the maxillary first molars and the mesial (M) roots of the mandibular first molars. Twenty-eight maxillary and 31 mandibular first molars were embedded, sectioned, stained, and observed at 30× magnification to evaluate the incidence and microscopic anatomy of the isthmus. The incidence of an isthmus 3 mm from the apex was 89.3% and 100% in the MB roots of the maxillary first molars and in the M roots of the mandibular first molars, respectively. The mean dentin thickness between the isthmus and the distal root surface was <1 mm at a distance of 3 mm from the apex in both types of roots. In this study, whenever two main canals were located in the MB roots of the maxillary first molars and in the M roots of the mandibular first molars, the likelihood of the presence of an isthmus increased. Therefore, clinicians should be aware of the thinnest dimensions in the distal surface of the MB roots of the maxillary first molars and the M roots of the mandibular first molars during nonsurgical and surgical root canal treatment.