This study explored primary school teachers' knowledge, attitude and practice of differentiated instruction. The target population of this study was primary school (Grades 1-4) teachers of the Amhara Region who were attending summer in-service diploma level training at Debre Markos College of Teacher Education in 2017 academic year. To this end, questionnaire and FGD were used as data collection instruments. The findings of the study revealed that there was a general level of understanding of differentiated instruction among primary school teachers. Though there was a seemingly adequate level of understanding of differentiated instruction by primary school teachers regarding the ways to support each group of students (i.e., fast learners, medium learners, and slow learners), teachers lacked knowledge of specific strategies to manage mixed ability classrooms in a way that engages each group of students during classroom hours simultaneously. The findings also indicated that there was a lower degree of implementation of differentiated instruction as compared to their level of understanding. It was also found that differentiation of content was the lowest practiced area. The data revealed that teachers were not regularly differentiating instruction in their classrooms due to lack of knowledge of specific strategies, the time constraints to prepare differentiated instructional lessons, and lack of relevant resources. Some teachers mentioned that large class size also obstructed their attempt of implementing differentiated instruction. They also do not usually have adequate opportunities to plan ahead and reflect on their work due to extremely high work load. To alleviate these problems, the researcher has forwarded relevant recommendations in the paper.