Abstract Ultrasonographic evaluation of polycystic ovarian morphology (PCOM) is part of the diagnostic evaluation of polycystic ovarian syndrome (PCOS). The relevance of PCOM as a diagnostic criterion for PCOS was reaffirmed in the most recent International Evidence-Based Guideline for the Assessment and Management of PCOS. However, there remains a lack of clarity regarding the best practices and specific ultrasonographic markers to define PCOM. We synthesized evidence on diagnostic test accuracy of ovarian ultrasound features to comprehensively determine the most suitable markers to diagnose PCOS. The primary outcome was diagnostic accuracy measures (e. g., thresholds, sensitivity, specificity) for PCOS using the following ovarian markers: follicle number per ovary (FNPO) or per single cross-section (FNPS), ovarian volume (OV), and stromal features. Databases of PubMed, Web of Science, Scopus, CINAHL, and CENTRAL were searched until 7 November 2021 to identify studies that compared ultrasound markers between women with PCOS to those without PCOS. Risk of bias and applicability assessment for diagnostic test accuracy studies were determined using the QUADAS-2 and QUADAS-C tool for a single index test or between multiple index tests, respectively. From a total of 1869 records initially identified, 22 studies were included and 18 (N=6,337; [n=2991 PCOS; n= 3346 Control]) were pooled for meta-analysis. FNPO was the most accurate diagnostic marker (sensitivity: 84%, CI: 81% to 87%; specificity: 91%, CI: 87% to 94%). OV and FNPS had similar but inferior pooled sensitivity (OV: 81%, CI: 75% to 86%; FNPS: 81%, CI: 70% to 89%) and specificity (OV: 79%, CI: 73% to 84%; FNPS: 83%, CI: 75% to 88%) compared to FNPO. Majority of studies had high risk of bias for patient selection and index test methodology across all markers. Subgroup analysis indicated that stratification based on age, body mass index (BMI), and previously proposed thresholds did not account for the heterogeneity in diagnostic accuracy observed across studies. For FNPO, studies that used a transducer frequency <8MHz or the Rotterdam criteria had improved positive likelihood ratio (+LR) and sensitivity, respectively. European studies had improved diagnostic accuracy (specificity, diagnostic odds ratio, +LR) compared to North American studies for FNPO, potentially due to BMI differences between PCOS populations (North America: 30.29 ± 0.61kg/m2, Europe: 27.62 ± 0.73kg/m2). Our findings support the use of FNPO as the gold standard in the ultrasonographic diagnosis of PCOS, with OV and FNPS as robust alternatives if total antral follicle counts cannot be obtained. These findings also identify avenues for future research to refine the ultrasonographic definition of PCOM for timely diagnosis of PCOS and investigations into phenotypic variations in pathogenesis and response to treatment in this condition (PROSPERO ID: CRD42021259118). Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.