The Spanish Female Sexual Function Index (FSFI) is a widely used scale to assess sexual dysfunction (SD), but its validation in Spanish women with multiple sclerosis (MS) has not been done. This cross-sectional multicenter study aimed to investigate the psychometric properties of the 19-item Spanish version of FSFI (svFSFI) in 137 women with MS and evaluate the prevalence of SD and its association with clinical and sociodemographic variables. Results showed excellent test-retest reliability and substantial-to-excellent internal consistency for the svFSFI. Convergent validity was significant in domain intercorrelations. Discriminant validity revealed differences in SD between women with high and low neurological disability, measured with Expanded Disability Status Scale (EDSS) scores. Exploratory factor analysis suggested a five-factor structure for the svFSFI. The prevalence of SD in the MS cohort was 42.6%, with 'desire' and 'arousal' being the most affected spheres. Only EDSS, fatigue, depression, and having a stable partner influenced the total svFSFI. The study confirmed the svFSFI as suitable for evaluating SD in Spanish women with MS. MS predominantly affects young women, making SD a frequent occurrence in this population. The study's strengths include a relatively large multicenter cohort and being the first validation of the svFSFI in women with MS. However, disbalanced patient numbers in EDSS categories and lack of responsiveness evaluation to intervention are limitations. Overall, the svFSFI is a reliable and valid questionnaire to assess sexual spheres in women with MS, revealing the prevalence of SD in this population.