This study examined specifically, the effect of item sequencing on the academic performance in mathematics among SS3 students in Calabar Education Zone of Cross River State. One null hypothesis formulated by the researchers offered guidance to the study. The study followed the quasi-experimental research design using two experimental and one control group. The stratified random sampling technique was adopted in selecting a sample of 474 SS3 students from a population of 9,768 students distributed across nine schools in Calabar Education Zone. The instrument utilized for information assortment was a mathematics achievement test (MAT). The null hypothesis was tested at .05 level of significance using Analysis of Covariance (ANCOVA). The results from the analysis revealed that test item sequencing significantly affects students’ academic performance in mathematics, with those who answered the easy-to-difficult arrangement performing significantly higher than those who answered difficult-to-easy arrangement and random. While those who answered difficult-to-easy arrangement performed the least in the mathematics achievement test. Based on these findings it was recommended among others that for better students’ academic performance in mathematics, the examination bodies should adopt the method of arranging test items in order of difficulty (simple to complex).