The need for measures of health-related quality of life (HRQOL) for clinical effectiveness research and for quality of care research, particularly for chronic diseases, is increasingly recognized. We assessed a measure of HRQOL for people with multiple sclerosis, a chronic neurological condition. We used the RAND 36-Item Health Survey 1.0 (aka SF-36) as a generic core measure, to enable comparisons of HRQOL of patients with multiple sclerosis to those of other patient populations and to the general population. To enhance comparisons within groups of multiple sclerosis patients, these items were supplemented with 18 additional items in the areas of health distress (four items), sexual function (four items), satisfaction with sexual function (one item), overall quality of life (two items), cognitive function (four items), energy (one item), pain (one item), and social function (one item). The final measure, the Multiple Sclerosis Quality of Life (MSQOL)-54 Instrument, contains 52 items distributed into 12 scales, and two single items. Internal consistency reliability estimates for the 12 multi-item scales ranged from 0.75 to 0.96 in a sample of 179 patients with multiple sclerosis. Test-retest intraclass correlation coefficients ranged from 0.66 to 0.96. Exploratory factor analysis confirmed two underlying dimensions of physical health and mental health. Construct validity was supported by significant associates between MSQOL-54 scales and degree of multiple sclerosis symptom severity in the prior year, level of ambulation, employment limitations due to health, admission to hospital in the previous year, and depressive symptoms.