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Aim To investigate the root canal anatomy of mandibular premolars in a Jordanian population. Methodology Nine hundred extracted mandibular premolars were examined. After the length of the teeth was measured, the presence of developmental grooves and furcated roots was noted. Following the preparation of access cavities, pulp tissue was removed and the canal systems were stained. The teeth were then rendered clear by demineralization and immersion in methyl salicylate. Cleared teeth were examined and the following features were evaluated: (i) type of root canals; (ii) presence and location of lateral canals; transverse anastomosis; (iii) location of apical foramina; and (iv) frequency of apical deltas. Results The mean lengths of first and second mandibular premolars were 22.6 mm (18–27.5 mm) and 22.2 mm (16–26.5 mm), respectively. Although the majority of the specimens corresponded to Vertucci’s classification scheme, analysis of this large data set revealed four additional root canal morphologies. Variable root canal morphologies were found in the mandibular first premolars; two separate apical foramina were found in 33% of the teeth with two canals, compared to 6.2% with one apical foramen. Teeth with three separate apical foramina were scarce (2.2%). The majority of the mandibular second premolars had a single canal; 72% of teeth possessed type I canal systems, whilst 22.8% of the roots had two canals with two separate apical foramina. Conclusions The prevalence of multiple canals in the investigated Jordanian mandibular premolars was high, especially for the second mandibular premolar, in comparison with previous studies performed on populations of different racial origin. [ABSTRACT FROM AUTHOR]