Hospital information systems (HIS) are often implemented to enhance the quality of care, as well as to improve the efficiency and safety of health care services. However, there are various barriers for their successful implementation. The aim of this paper is to prioritize these barriers. This research is a cross sectional analytic-descriptive study. The study populations were hospital managers, IT department administrators, and clinical supervisors at the academic and non-academic hospitals of two cities in Iran. The data was collected by a questionnaire that its content validity was confirmed by three specialists. Its reliability was confirmed using Cronbach's alpha (α = 0.78). Questionnaire contained five dimensions and 39 implementation barriers. The collected data was analyzed by descriptive and analytical statistics using the Kendall Rank Correlation Coefficient and Chi2 tests. The findings of the study revealed that lack of powerful information networks, error in data entry, technical problems related to system design, lack of organizational training, lack of users' knowledge about system and working with it, and negative attitudes of providers and patients toward systems are the most important barriers of HIS implementation. Prioritizing of these barriers helps policy makers to decide what to do when planning for HIS utilization.