Poor neck and shoulder postures have been suggested to be a cause of neck and shoulder pain in computer workers. The present study aimed to evaluate and compare the head, neck and shoulder postures of office workers with and without symptoms in these regions, in their actual work environments. The two all female subject groups reported significantly different discomfort scores across five trials repeated in a single working day. The results of repeated video capture and two-dimensional motion analysis showed that there were trends for increased head tilt and neck flexion postures in the symptomatic subjects (n = 8), compared to the asymptomatic subjects (n = 8). Symptomatic subjects also tended to have more protracted acromions compared with asymptomatic subjects and showed greater movement excursions in the head segment and the acromion. All subjects demonstrated an approximately 10% increase in forward head posture from their relaxed sitting postures when working with the computer display, but there were no significant changes in posture as a result of time-at-work.