The aim of this paper is to examine the attitudes of Croatian teachers of English as a foreign language (EFL) towards the role and use of differentiated instruction in the EFL classroom. Using an online questionnaire an investigation was conducted into how frequently the participants encountered specific classroom situations or performed specific activities when teaching English to mixed-ability groups, and which type of students were most likely to receive differentiated instruction and attract their attention. Also, the familiarity of EFL teachers with specific differentiated instruction strategies was explored (Tomlinson 2001), as well as their opinion on the strategies they used in the classroom, the challenges they faced when using differentiated instruction and the factors that caused mixed-ability classrooms. The findings in this study reveal a gap between teachers’ perceptions of differentiated instruction at the theoretical level and their use in actual EFL practice. Teachers seem to be aware of student differences and claim that they use differentiated instruction, in particular with weaker students. They also maintain that they are familiar with specific differentiated instruction teaching strategies; however, when reference is made to their classroom practice, the participants seem to demonstrate inadequate knowledge and tend to use a rather limited repertoire of teaching strategies. The findings also reveal that the participants have a negative attitude towards mixed-ability classes, which they believe have an adverse effect on teaching and learning. It will be argued that teachers would benefit from more training in the field so as to be able to put theory into practice in the EFL classroom.