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Professionalism requires that language teachers be assessment literate so as to assess students’ performance more effectively. However, assessment literacy (AL) has remained a relatively unexplored area. Given the centrality of AL in educational settings, in the present study, we identified the factors constituting AL among university instructors and examined the ways English Language Instructors (ELIs) and Content Instructors (CIs) differed on AL. A researcher-made, 50-item questionnaire was constructed and administered to both groups: ELIs (N = 155) and CIs (N = 155). A follow-up interview was conducted to validate the findings. IBM SPSS (version 21) was used to analyse the data quantitatively. Results of exploratory factor analysis showed that AL included three factors: theoretical dimension of testing, test construction and analysis, and statistical knowledge. Further, results revealed statistically significant differences between ELIs and CIs in AL. Qualitative results showed that the differences were primarily related to the amount of training in assessment, methods of evaluation, purpose of assessment, and familiarity with psychometric properties of tests. Building on these findings, we discuss implications for teachers’ professional development.