Definitive diagnosis of endometriosis is established by histologic confirmation in tissue from surgically visualized lesions; however, the diagnostic sensitivity of this approach varies widely. We hypothesized that incomplete tissue block sampling may contribute to false-negative diagnosis, particularly if the focus of endometriosis in the tissue section is scant. This study defined the diagnostic value of deeper level tissue sections in cases in which none of the specimen parts contained endometriosis on the initial tissue sections, using the World Health Organization essential criteria for diagnosis of endometriosis (presence of endometrial glands and endometrial stroma). Among 135 patients who underwent surgery for suspected endometriosis by a single surgeon at an academic institution from 2015 to 2019, the initial tissue sections resulted in a diagnosis of endometriosis in 73.3% (99/135), at an average diagnostic yield of 5.9 slides per diagnosis of endometriosis. An additional 9 patients were diagnosed with endometriosis by deeper level tissue sections, increasing the diagnostic rate to 80% (108/135). This 6.7% gain in the diagnostic rate came at an increase in resource utilization, with an overall overage diagnostic yield of 9.8 slides per diagnosis of endometriosis. Overall, 8.3% of patients had a false-negative diagnosis on the initial tissue sections. When extrapolated to a population level, the number of patients potentially affected by this source of false-negative diagnosis and the implications for patients merit consideration of the use of deeper level sections if none of the initial sections of any of the specimens contains endometriosis.