This study sought to examine teacher educators‘ perceptions, self-efficacy, feeling of preparedness and practices of differentiated instruction in teacher education colleges of Amhara region. For this purpose, a mixed methods research approach with concurrent mixed methods design was used. The study generated data from both primary and secondary sources. Out of the ten Teacher Education Colleges (CTEs) of the Amhara Region, randomly selected six CTEs (Gondar, Debre Markos, Enjibara, Finote Selam, Dessie, and Begemdir) were considered as sample sites. From the sample CTEs, 400 teacher educators were selected using a stratified sampling technique. As secondary sources, various documents such as course plans, and assessment guides were employed. The main data gathering instruments were: questionnaire, interview, observation, and document analysis. The data collected were analyzed quantitatively using descriptive and inferential statistics (frequencies, percentages, mean, standard deviations, one sample t-test, independent samples t-test, one-way ANOVA, and multiple regression) and qualitatively using case-based thematic analysis, descriptions, and narrations. The findings revealed that teacher educators in CTEs in the Amhara Region have a positive perception of differentiated instruction (DI) to address learner diversity. They perceived that their students differ greatly in their readiness, learning profile, educational background, and interest, and these differences in students‘ characteristics have an impact on the contents they teach, the pedagogy they employ, and the assessment techniques they use. On the other hand, teacher educators were less efficacious in implementing DI. Most of them experienced a certain degree of fear and uncertainty in their capacity to implement DI. Besides, teacher educators felt that they were poorly prepared to differentiate instruction. Teacher educators‘ level of implementation of DI in college classrooms was also low. However, there is a significant positive relationship between teacher educators‘ perception of DI, their feeling of preparedness, and their self-efficacy beliefs with DI practices, which in turn proved that teacher educators‘ perception, self-efficacy and feeling of preparedness have a major influence and can predict their practice of differentiated instruction in the college classrooms. The major barriers affecting the implementation of DI were: insufficient time, rigidity in the school curriculum, high workloads, big class sizes, low motivation and commitment among teacher educators, and inadequate knowledge and abilities in DI. Thus, it is suggested that the Amhara regional education bureau and college officials should organize training that will provide instructors with sufficient opportunities to learn how to differentiate instruction, strengthen their DI self-efficacy, and help them feel well-prepared for differentiation.