The sphenoid ostium (SO) provides a natural portal for entering the sphenoid sinus and beyond up to the skull base. It is not always easy to locate the ostium during the endoscopic approach. The present study was designed to establish readily identifiable anatomical landmarks for locating the sphenoid ostium. Cadaveric dissection was performed in 30 hemisections of head and neck and various measurements were taken from fixed anatomical landmarks in the nasal cavity to the sphenoid ostium. The size, shape and position of sphenoid ostium were determined in relation to the anterior wall of the sphenoid sinus and the superior turbinate. The mean distance from the supero-lateral angle of the posterior choana to the SO was found to be 21.21 ± 6.02 mm. The mean distance of the SO from the midline was 4.85 ± 2.89 mm. In all the specimens, the SO was situated within 1 cm of the midline. The mean distance between the inferior end of the SO and the postero-inferior edge of the superior turbinate was 8.03 ± 3.52 mm. The SO was present on an average distance of 55.1 ± 3.54 mm from the limen nasi. In 93.3 % of the specimens, the SO was situated between 5 and 6 cm of the inferior end of the limen nasi. The angle between the anterior nasal spine and the SO was found to be remarkably constant. In 93.3 % of the specimens, it was from 25° to 30°. The sphenoid ostium could be localized medial to the superior turbinate between 1.5 and 3 cm above the supero-lateral angle of the posterior choana, within 1 cm of the midline and within 1 cm of the postero-inferior edge of the superior turbinate.