<p>In this study, the root canal morphology, radicular groove (RG) presence, and canal separation level (FCAP) in mandibular premolars of a Turkish population were evaluated using cone-beam computed tomography (CBCT). CBCT images of 3594 mandibular premolars from 402 males and 564 females were analyzed. Root canal morphology was classified using Vertucci’s system and Ahmed’s classification. RG presence and location and FCAP levels were also recorded. The most common configuration in Ahmed’s classification was ¹TN¹, followed by ¹TN^1–2. Vertucci Configuration I was the most frequent, with Configuration V being the second most common. Some canal configurations showed significant sex-related differences (<i>p</i> &lt; 0.01), whereas no age-related differences were found. Bilateral symmetry was generally high. RGs were more common in first premolars and were usually located in the middle third. RG presence was associated with Vertucci Configuration V and with the ¹TN^1–2 and ²TNB¹L¹ configurations. FCAP was most frequently located in the middle third of the root. The root canal morphology of mandibular premolars exhibited a high degree of bilateral symmetry, while complex configurations were associated with the presence of RGs and the level of canal separation (FCAP). These findings have clinical relevance for planning canal shaping and irrigation strategies in endodontic treatment.</p>