Importance: Safe entry into sphenoid sinus is critical in endoscopic sinus and skull base surgery. A number of surgical landmarks have been used to identify the sphenoid sinus ostium during endoscopic endonasal surgery with variable reliability and intraoperative feasibility. Objective: To determine if the posterior wall of the maxillary sinus is a reliable landmark to determine the depth of the sphenoid sinus ostium during anterior to posterior dissection. Design, Setting, and Participants: Prospective study of adult patients undergoing endoscopic sinus surgery between August 2016 and September 2017. Measurements were made intraoperatively between the depth of the posterior maxillary sinus wall and sphenoid sinus ostium. Main Outcomes and Measures: The primary measurement is the distance between the depth of the posterior maxillary sinus wall and sphenoid sinus ostium. Additional data points included age, gender, surgical indication, and primary versus revision endoscopic sinus surgery. Results: Forty-five patients (38% male, 62% female) with an average age of 56 were enrolled, resulting in 88 operated sides. The average distance between the depth of the posterior wall of the maxillary sinus and the sphenoid ostium was 1.5 mm ± 1.4 mm. The most common position of the sphenoid sinus ostium was posterior to the level of the posterior maxillary sinus wall (54%), followed by same level (23%) and anterior (23%). There was no significant difference between different disease states ( P = .75) and between primary and revision cases ( P = .13). Conclusions and Relevance: The posterior wall of the maxillary sinus serves as an adjunctive intraoperative landmark to determine the depth of the sphenoid sinus ostium. While the posterior wall of the maxillary sinus approximates the depth of the sphenoid sinus ostium, the relative position is variable and can be anterior or posterior.