OBJECTIVE: Identification of anatomical landmarks is essential for interpretation of video fluoroscopic swallow studies (VFSS). This investigation sought to confirm the location of essential laryngeal landmarks and determine clinician accuracy in structure identification on VFSS. METHODS: A single human cadaver was used to generate unmarked standard lateral and anterior-posterior (AP) fluoroscopic images. Essential laryngeal structures (e.g., true vocal fold, arytenoid) were directly identified using a guidewire placed through an endoscope while obtaining corresponding marked fluoroscopic images. Licensed clinicians (speech-language pathologists [SLP], laryngologists) and trainees (otolaryngology residents, SLP clinical fellows [CF]) identified 18 structures (9 lateral, 9 AP) on unmarked images. Answers were compared to corresponding marked images. The percentage of accurate identification was calculated for each clinician and then compared between groups using t-tests. RESULTS: Twenty-four individuals (10 SLPs, 1 CF, 9 residents, 4 laryngologists) from six institutions completed structure identification. Mean overall accuracy was 41.7 ± 13.0% (range 18.8-68.8%). There were no significant differences in mean overall accuracy between trainees (41.9 ± 12.9%) and clinicians (42.0 ± 13.1%), p = .97, or between SLPs (45.5 ± 12.8%) and physicians (38.9 ± 12.3%), p = .22. On average, participants were significantly more accurate identifying structures on lateral view (53.1 ± 16.1%) than AP (27.3 ± 22.8%), p < .001. Less than half of participants accurately identified the laryngeal ventricle, cricoid, epiglottic petiole, and the anterior commissure on lateral view. CONCLUSIONS: The ability of certified clinicians and trainees to correctly identify essential anatomic landmarks on swallowing fluoroscopy may be poor. Future work is needed to identify how we can train clinicians on more accurate identification of essential anatomic structures on swallowing fluoroscopy.Level of Evidence: NA.