Background: Clinical airway screening tests intend to predict difficult airways, but none have a high predictive value. Recent systematic reviews correlate ultrasound with difficult laryngoscopy. This study aimed primarily to correlate ultrasound measurements of anatomical upper airway structures in the sniffing position with difficult direct laryngoscopy. The secondary aim was to observe gender-based differences. Methods: This prospective, cross-sectional, single-center observational study included 209 patients requiring general anesthesia for elective surgery. Preoperatively, we performed six clinical airway assessments and three ultrasound measurements, which were the Distance from Skin to the Hyoid Bone (DSHB), to the Epiglottis (DSE), and to the anterior commissure of the vocal cords (DSAC) in a sniffing position. Benumof's criteria for the ¿best view at the first attempt¿ for direct laryngoscopy assessed the difficulty of laryngoscopy. Results: The distance from skin to the epiglottis was the best predictor of direct difficult laryngoscopy (defined as Cormack-Lehane grade ¿ 2b) with a minimum thickness cut-off at 2.70 ± 0.19 cm (sensitivity 91.3%; specificity 96.9%). The skin to the hyoid bone distance cut-off was 1.41 ± 0.30 cm with moderate correlation (sensitivity 80.4%; specificity 60.1%). No correlation was found for the distance to the anterior commissure of the vocal cords. In women compared to men, the skin to the epiglottis distance was more sensitive (92.3% vs. 90.9%) and specific (98.8% vs. 95.2%). Conclusions: DSE in the sniffing position is the most reliable parameter for preoperative airway ultrasound assessment in the Caucasian population, with higher sensitivity and specificity in women, and might be considered as an independent predictor for direct difficult laryngoscopy.