Students respond to classroom activities and achievement outcomes with a variety of emotions that can impact student success. One emotion students experience is anxiety, which can negatively impact student performance and persistence. This study investigated what types of classroom anxiety were related to student performance in the course and persistence in the major. Students in introductory biology classes self-reported their general class, test, communication, and social anxiety; perceived course difficulty; intention to stay in the major; and demographic variables. Final course grades were acquired from instructors. An increase in perception of course difficulty from the beginning to the end of the semester was significantly associated with lower final course grades (N = 337), particularly for females, non-Caucasians, and students who took fewer Advanced Placement (AP) courses. An increase in communication anxiety slightly increased performance. Higher general class anxiety at the beginning of the semester was associated with intention to leave the major (N = 122) at the end of the semester, particularly for females. Females, freshmen, and those with fewer AP courses reported higher general class anxiety and perceived course difficulty. Future research should identify which factors differentially impact student anxiety levels and perceived difficulty and explore coping strategies for students.