In response to the mandates of No Child Left Behind, (NCLB), educators across the country struggle to close the gaps between males and females. Some of the physiological differences existing between the male and female brain suggest support for single-gender instruction, which is on the rise within this country as well as other parts of the world. Using the theoretical framework based on brain research, the purpose of this quantitative study was to compare the effect of single-gender instruction on assessment results in Palmetto Assessments of State Standards (PASS) math and reading scores of public middle single-gender schools to mixed-gender schools in South Carolina. This was a causal-comparative research study because the data used came from the South Carolina's educational website with the focus on individual school report cards of the 78 middle schools which were used in this study. Mean scores from the Palmetto Assessments of State Standards (PASS) performance statistics spreadsheet in math and reading were used in determining achievement levels of single-gender and heterogeneous instruction. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://bibliotheek.ehb.be:2222/en-US/products/dissertations/individuals.shtml.]