This nationwide study of 532 female school district superintendents, the largest such sample to date, offers compelling evidence that unconscious gender bias exists on the job and further inhibits equitable female representation in the superintendency. A modified version of Tran et al.'s (2019) Perceived Subtle Gender Bias Index (PSGBI), was confirmed as a valid instrument for assessing female superintendents' perceptions of unconscious gender bias and produced the same four factors as the original PSGBI. Findings support Joan Acker's (1990) theoretical assertion that gender inequality is deeply embedded within organizational structures, patterns, and processes. Respondents reported that gender bias occurs more frequently than the profession acknowledges and suggested that it derives primarily from sources other than superintendents' colleagues. Recommendations offered.