Abstract Background Restless legs syndrome (RLS) is a frequent neurological disorder which is presented in idiopathic and secondary form. Idiopathic RLS is associated with common genetic variants in four chromosomal regions. Recently, multiple sclerosis (MS) was identified as a common cause for secondary RLS. The aim of our study was to evaluate the prevalence of RLS among Czech patients with MS and to further analyze the impact of known genetic risk factors for RLS in patients with MS. Methods Each patient underwent a semi-structured interview. A patient was considered to be affected by RLS if all four standard criteria had ever been met in their lifetime. The sample was genotyped using 12 single nucleotide polymorphisms within the four genomic regions, which were selected according to the results of previous genome-wide association studies. Results A total of 765 subjects with MS were included in the study and the diagnosis of RLS was confirmed in 245 subjects (32.1%, 95%CI 28.7–35.4%). The genetic association study included 642 subjects; 203 MS patients with RLS were compared to 438 MS patients without RLS. No significant association with MEIS 1, BTBD9, and PTPRD gene variants was found despite sufficient statistical power for the first two loci. There was a trend for association with the MAP2K5/SCOR1 gene – the best model for the risk allele was the recessive one (p nominal=0.0029, p corrected for four loci and two models=0.023, odds ratio=1.60). Conclusion We confirmed that RLS prevalence was high in patients with multiple sclerosis, but this form did not share all genetic risk variants with idiopathic RLS.