Background: For the case of multiple sclerosis, research on gender differences from a health economics perspective has not received much attention. However, cost-of-illness analyses can provide valuable information about the diverse impact of the disease and thus help decision-makers to allocate scarce resources. The aim of this study was to describe healthcare resource use and associated societal costs from a gender perspective. In particular, we aimed to identify how resource utilization potentially differs in certain cost components between men and women. Methods: Clinical and economic data were extracted from two prospective, multicentre, non-interventional, observational studies in Germany. Information on health resource use was obtained from all patients on a quarterly basis using a validated questionnaire. Cost analyses were conducted from the societal perspective including all direct (healthcare-related) and indirect (work-related) costs, regardless of who bears them. Gender-related differences were analysed by a multivariable generalized linear model with a negative binomial distribution and log link function due to the right-skewed distribution pattern of cost data. In addition, costs for men and women were descriptively analysed within subgroups of two-year disease activity. Results: In total, 2095 patients (women-to-men ratio of 2.7:1) presented a mean age of 41.85 years and a median Expanded Disability Status Scale of 2 (interquartile range 1–3.5) ( p > 0.30 for gender-related differences). Women and men did not statistically differ in total quarterly costs (€2329 ± €2570 versus €2361 ± €2612). For both, costs were higher with advancing disease severity and indirect costs were the main societal cost driver. Regarding healthcare-related resources, women incurred higher costs for ambulant consultations [incidence rate ratio (IRR) 1.16, confidence interval (CI) 1.04–1.31], complementary medicine (IRR 2.41, CI 1.14–5.06), medical consumables (IRR 2.53, CI 1.69–3.79) and informal care (IRR 2.79, CI 1.56–5.01). Among indirect costs, we found higher costs for men for presenteeism (IRR 0.62; CI 0.53–0.72) and higher costs for women for disability pension (IRR 1.62; CI 1.23–2.13). Conclusions: Multiple sclerosis poses a significant economic burden on patients, families and society. While the total economic burden did not differ between male and female patients, we found gender differences in specific cost items that are similar to those in the wider non-MS population.