Objective: Pattern separation (PS) is the ability to represent similar experiences as separate, nonoverlapping representations. It is usually assessed via the Mnemonic Similarity Task -Object Version (MST-O) which, however, assesses PS performance without taking behavioral context discrimination into account, since it is based on pictures of everyday simple objects on a white background. We here present a validation study for a new task, the Mnemonic Similarity Task -Context Version (MST-C), which is designed to measure PS while taking behavioral context discrimination into account by using real-life context photographs. Methods: Fifty healthy subjects underwent the two MST tasks to assess convergent evidence. Instruments assessing memory and attention were also administered to study discriminant evidence. The test-retest reliability of MST-C was analyzed. Results: Weak evidence supports convergent validity between the MST-C task and the MST-O as measures of PS (r s = 0.464; p o 0.01); PS performance assessed via the MST-C did not correlate with memory or attention; a moderate test-retest reliability was found (r s = 0.595; p o 0.01). Conclusion:The MST-C seems useful for assessing PS performance conceptualized as the ability to discriminate complex and realistic spatial contexts. Future studies are welcome to evaluate the validity of the MST-C task as a measure of PS in clinical populations.