Background High‐grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer, and it is often associated with ascites at presentation. The objective of this study was to evaluate the accuracy of cytopathology to identify immunophenotypic features of HGSC and BRCA1/2 mutations from ascites. Methods The study included 45 patients with histologically confirmed primary HGSC and malignant ascites. Immunocytochemistry (ICC) staining for PAX8, WT1, P53, P16, and Ki‐67 was performed on cytospins and cytoblocks prepared from ascites. Next‐generation sequencing (NGS) was used to detect germline/somatic BRCA1/2 mutations in the ascites. Both ICC and NGS results were compared with immunohistochemistry (IHC) and NGS results from tissue blocks of primary tumor. Cronbach α and χ2 statistics, respectively, were used. Results ICC/IHC results for PAX8, WT1, P53, and P16 showed good reliability between cytospins, cytoblocks, and tissue blocks (α > 0.75), whereas poor reliability and significant differences were observed for Ki‐67 between ascites and tissue blocks (α < 0.26; p < .001 Kruskal–Wallis). For germline BRCA1/2 mutations, 100% concordance was confirmed, but only 14% concordance was confirmed for somatic mutations. Conclusions These results demonstrate that cytopathology is an accurate method for identifying immunophenotypic features of HGSC and detecting germline BRCA1/2 mutations from ascites. However, further investigation is required for assessing the proliferation activity of HGSC in ascites and for detecting somatic BRCA1/2 mutations. Cytopathological assessment of immunophenotypic features and germline BRCA1/2 mutations of high‐grade serous carcinoma from ascites is comparable to histological assessment of the primary tumor. This approach is valuable when optimal surgical debulking cannot be performed.