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Background: Stressful situation are among the challenging experiences especially in clinical setting. The aim of this study was to compare the level of work-related stress among nurses working in Intensive Care Units (ICUs), General ward and Emergency setting. Methods: This cross-sectional study was conducted at different private tertiary hospitals of Karachi in 2017-2018. Two stage sampling technique including quota and convenient sampling technique was used to collect the data. A total of 225 participants were recruited. Pre-validated questionnaire was used which contained different stress related categories. Midwives, nursing assistants, part time nurses and nurses employed at management positions were excluded from the study. Data were presented as frequency and percentage and mean and standard deviation. The chi-square test was used for examine association of categorical variables. The SPSS used for data analysis. Results: The mean age of participants was 26.5±2.4 years. Stress among nurses working in emergency department was found to be highest in all categories. Management related stress was 77.3% and 69.3% due to criticism by supervisor and inadequate support by supervisor, respectively. Patient related stress was 72% and 64% due to health risk posed by patient contact and communication about death to patient. About 81.3% of the nurses of emergency department reported high level of stress due to covering work for another employee followed by 66.7% related to working overtime. Moreover, 86.7% and 62.7% of participants had high level of stress due to inadequate salary and lack of recognition. Conclusion: The study concludes that the level of stress varies within different departments in the hospitals. There are several work-related stressors that found more in Emergency department than ICU or General ward.