In gender differential item functioning (DIF) research it is assumed that all members of a gender group have similar item response patterns and therefore generalizations from group level to subgroup and individual levels can be made accurately. However DIF items do not necessarily disadvantage every member of a gender group to the same degree, indicating existence of heterogeneity of response patterns within gender groups. In this article the impact of heterogeneity within gender groups on DIF investigations was investigated. Specifically, it was examined whether DIF results varied when comparing males versus females, gender × socioeconomic status subgroups and latent classes of gender. DIF analyses were conducted on reading achievement data from the Canadian sample of the Programme of International Student Assessment 2009. Results indicated considerable heterogeneity within males and females and DIF results were found to vary when heterogeneity was taken into account versus when it was not.