Aims: To examine the dimensionality, internal consistency, and construct validity of a new physical workload questionnaire in employees with musculoskeletal complaints. Methods: Factor analysis was applied to the responses in three study populations with musculoskeletal disorders (n = 406, 300, and 557) on 26 items related to physical workload. The internal consistency of the resulting subscales was examined. It was hypothesised that physical workload would vary among different occupational groups. The occupations of all subjects were classified into four groups on the basis of expected workload (heavy physical load; long lasting postures and repetitive movements; both; no physical load). Construct validity of the subscales created was tested by comparing the subscale scores among these occupational groups. Results: The pattern of the factor loadings of items was almost identical for the three study populations. Two interpretable factors were found: items related to heavy physical workload loaded highly on the first factor, and items related to static postures or repetitive work loaded highly on the second factor. The first constructed subscale "heavy physical work" had a Cronbach's α of 0.92 to 0.93 and the second subscale "long lasting postures and repetitive movements", of 0.86 to 0.87. Six of eight hypotheses regarding the construct validity of the subscales were confirmed. Conclusions: The results support the internal structure, internal consistency, and validity of the new physical workload questionnaire. Testing this questionnaire in non-symptomatic employees and comparing its performance with objective assessments of physical workload are important next steps in the validation process.