The first and most crucial step towards developing a sustainable curriculum for instructors teaching English for Specific Academic Purposes (ESAP) is a needs analysis. Therefore, the main aim of conducting this study was to investigate the in-service needs of language instructors and content specialists teaching ESAP and to spot the differences between the needs of these two groups in order to provide them with systematic treatments in ESAP teacher training programs. This mixed method study was designed on a qualitative-quantitative survey basis using a questionnaire, a semi-structured interview, and an observation checklist. The analysis of the data collected from 50 content specialists and 50 language instructors completing the questionnaires reveals that there is a significant difference between the in-service needs of these two groups, that is, language instructors desire more to be trained in an in-service ESAP teaching training program in terms of professional, procedural and personal needs. Furthermore, the results of the data obtained from the semi-structured interview and the observation of 20 of the above-mentioned instructors (i.e., 10 content specialists and 10 language instructors) indicate that language instructors have more difficulty selecting suitable materials, suffer more from low income, attitudinal difficulties and backwash effect compared to their counterparts teaching ESAP courses. It can be inferred that the results of the present study can sufficiently help the researchers to embark on an in-service teacher training program both for ESAP content specialists and language instructors based on their specific needs in the ESAP context.