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Differential Item Functioning (DIF) analysis is a key element in evaluating educational test fairness and validity. One of the frequently cited sources of construct-irrelevant variance is gender which has an important role in the university entrance exam; therefore, it causes bias and consequently undermines test validity. The present study aims at investigating the presence of DIF in terms of gender in a high stakes language proficiency test in Iran, the National University Entrance Exam for Foreign Languages (NUEEFL). The participants’ responses (N = 5000) were selected randomly from a pool of examinees who had taken the NUEEFL in 2015. The results displayed DIF between male and female test takers. Hence, on the basis of the findings, it is concluded that the NUEEFL test scores are not free of construct-irrelevant variance and the overall fairness of the test is not confirmed. Also, both Rasch assumptions (i.e., unidimensionality and local independence) are hold in the present research.