The measurement tool not measuring the specific construct has a validity problem. Individuals based on the results obtained from this type of tool should not be evaluated. The purpose of this study was to examine the differentiated item functioning and item bias of mathematics items in the Programme for International Student Achievement 2012 assessment for gender using two-level hierarchical generalized linear model, logistic regression and experts' opinions. Also differentiated item functioning sources (anxiety, interest and self-efficacy) at student level were tested. The current study was created under take into account of quantitative and qualitative methods. It was conducted with 1458 students selected from 166 schools of Turkey sample. The results reveal that hierarchical generalized linear models approach is more conservative than logistic regression approach. When the student level variables were added to the model as potential sources, differentiated item functioning did not disappear for the three items. Also half of the experts argued that the items identified as in favor of boys are biased. Statements in the items and the context were given as the reasons for this bias.