Objective: Musculoskeletal discomfort is common in people working in poor ergonomic designs with prolonged poor posture. The main purpose of this study was to compare the postural discomfort among right and left-handed university students of Rawalpindi/Islamabad. Methodology: A cross-sectional survey was carried out using standardized Nordic Musculoskeletal Discomfort Questionnaire. Samples were collected by simple convenient sampling technique from 221 students (53.8% females and 46.2% Males) comprising of 110 left and 111 right-handed University students. Results: The comparison of percentages among left and right-handed participants at hip, knee and ankle were as follows: hip (left-handed 19% and 5.42% right-handed), knee (left-handed 17.19%, right-handed 9.04%), ankle (left-handed 9.50%, right-handed 8.14%). The prevalence of chronic musculoskeletal discomfort was significant in left-handed participants as compared to right-handed participants. The highest frequency of discomfort was found in the neck and shoulder region of left-handed participants. Chi square test showed a significant difference in frequencies of discomfort in left and right-handed participants (p<0.01), whereas there was as such no significance in ankle region (p>0.05). Conclusion: Postural discomfort was more prevalent among left-handed university students as compare to right-handed students. The highest frequency of discomfort was found in the neck and shoulder region of left-handed participants. Further research and attention should be directed towards the relation of handedness with musculoskeletal discomfort and factors influencing postural discomfort in left-handed students.