The study identifies the most important challenges facing general education male and female teachers in applying the differentiated instruction strategy in different stages of education in the Eastern Province in the Kingdom of Saudi Arabia. To achieve this, the researcher designed a questionnaire consisting of 47 paragraphs on five axes. It was distributed to 275 male and female teachers in Dammam city, after having verified its validity and stability, The results indicate that the degree of the challenge of using the differentiated instruction strategy was medium overall. The challenges related to students were ranked first, followed by challenges related to the school environment, then the nature of the differentiated instruction, challenges related to teachers, and, finally, challenges related to study courses. However, the results were not statistically significant for the variable of the educational stage (primary, intermediate, and secondary), although there were significant differences for the gender variable in favour of males. The study recommends creating a school environment that supports the use of a differentiated instruction strategy and holding workshops for teachers to train them in differentiated instruction activities.