We report the development of a simplified procedure for restriction fragment length polymorphism (RFLP) analysis of mushrooms. We have adapted standard molecular techniques to be amenable to an undergraduate laboratory setting in order to allow students to explore basic questions about fungal diversity and relatedness among mushroom species. The streamlined protocols allowed students to practice important molecular techniques within the context of self-designed investigative projects. This laboratory experience provided opportunities for students to practice strategies for examining molecular diversity among species.