This study, which is entitled, “Elizabeth I and the 1559 Act of Uniformity: A Study of the Impact of Gender Roles and Religious Conflict” analyzes the impact of 16th century gender roles and religious conflict to explain the decision of Elizabeth I, Queen of England from 1558 – 1603, to champion the passage of the Act of Uniformity through Parliament in 1559. Through the analysis of primary sources, specifically Elizabeth’s letters from her childhood through the Act’s passing in 1559, an understanding of these influences on Elizabeth is developed which illuminates important turning points in her life and the subsequent development of her personal desire to mitigate religious conflict in England and to bring unity to her people. The analysis was conducted through the use of historical analysis of primary sources in combination with the use of Narrative Thematic Analysis in order to discover themes within the sources. The themes which emerged then offered insight into Elizabeth’s personal development and her decisions regarding the Act of Uniformity. The focus of this dissertation is guided by the context of 16th century gender roles and the 16th century Protestant Reformation which ultimately laid the foundation for Elizabeth’s birth and directly influenced her education as well as religious and personal development. The impact of gender roles and the expectations placed upon Elizabeth is intertwined with the subsequent religious conflict Elizabeth witnessed in England from her birth. The results focus on illustrating areas of conflict in the 16th century and how each area of conflict is relevant to comprehend if there is to be success in altering the path of both gender conflict and religious conflict in the modern era.