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Stress among health care staff often resulting from high expectations coupled with insufficient time, skills and/or social support at work. This lead to severe distress, burnout or physical illness, decrease in quality of life and service provision. The study was aimed to determine the prevalence and associated factors of stress among assistant medical officers in Ministry of Health hospitals in Kelantan and Terengganu, Malaysia. A cross-sectional study was conducted on 194 randomly selected assistant medical officers in Ministry of Health hospitals in Kelantan and Terengganu. The questionnaire was a self-administered questionnaire which required responses on socio-demographic data, Malay Version DASS-42 and Job Content Questionnaires (JCQ). Salivary cortisol level was measured using Expanded Range High Sensitivity Salivary Cortisol Enzyme Immunoassay Kit by Salimetrics®. The prevalence of stress was 13.7%. Decision latitude, psychological job demand, job insecurity and total physical hazards were the significant associated factors of stress. Salivary cortisol was significantly higher among stressed compared to non-stressed assistant medical officers.