Students who lack metacognitive skills can struggle with the learning process. To be effective learners, students should recognize what they know and what they do not know. This study examines the relationship between students' perception of their knowledge and determined knowledge in an upper-level biology course utilizing a pre/posttest approach. Significant differences in students' perception of their knowledge and their determined knowledge exist at the beginning (pretest) and end (posttest) of the course. Alignment between student perception and determined knowledge was significantly more accurate on the posttest compared with the pretest. Students whose determined knowledge was in the upper quartile had significantly better alignment between their perception and determined knowledge on the pre- and posttest than students in the lower quartile. No difference exists between how students perceived their knowledge between upper-and lower-quartile students. There was a significant difference in alignment of perception and determined knowledge between males and females on the posttest, with females being more accurate in their perception of knowledge. This study provides evidence of discrepancies that exist between what students perceive they know and what they actually know.