The purpose of this study was to investigate the factors that impact teachers’ preparedness for and implementation of single-gender instructional strategies and culturally relevant pedagogy with African-American girls in single-gender classrooms. Three Single-Gender Academies were included in the study. Twenty participants responded to a teacher survey, nine teachers and two administrators were interviewed, and lesson plans from six of the teachers interviewed were also collected. Descriptive statistics, regression tests and Pearson’s Correlations were used to analyze quantitative data and displayed a significant relationship between professional development and teacher preparedness for and implementation of single-gender instructional strategies and culturally relevant pedagogy. A significant relationship also existed between teacher preparation program and teacher preparedness for implementing single-gender instructional strategies. No significant relationship was found between teacher demographics and teacher preparedness for and implementation of single-gender instructional strategies and culturally relevant pedagogy. Analysis of qualitative data revealed that certain teacher demographics did impact teacher preparedness for and implementation of single-gender instructional strategies and culturally relevant pedagogy for the teachers in this research investigation. Qualitative analysis of data provided evidence to support the quantitative findings about professional development and single-gender instructional strategies and culturally relevant pedagogy with African-American girls in single-gender classrooms. Moreover, analysis of qualitative data revealed that specific behaviors of administrators influenced teacher preparedness for and implementation of single-gender instructional strategies and culturally relevant pedagogy. Results of this research investigation provide implications and recommendations for school districts that incorporate single-gender classroom environments, faculty at Educational Departments at local universities and alternative teacher preparation programs and administrators that lead these schools.