Background: To differentiate five formulations of Interferon Beta for the treatment of multiple sclerosis (MS) in clinical practice, by analysing persistence, adherence, healthcare resource utilisation and costs at population level. Methods: In this population-based study, we included individuals with MS living in the Campania Region of Italy from 2015 to 2017, on treatment with intramuscular Interferon Beta-1a (Avonex® = 618), subcutaneous pegylated Interferon Beta-1a (Plegridy® = 259), subcutaneous Interferon Beta-1a (Rebif® = 1220), and subcutaneous Interferon Beta-1b (Betaferon® = 348; and Extavia® = 69). We recorded healthcare resource utilisation from administrative databases (hospital discharges, drug prescriptions, MS-related outpatients), and derived costs from the Regional formulary. We classified hospital admissions into MS-related and non-MS-related. Persistence (time to switch to other disease modifying treatments (DMTs)), and adherence (medication possession ratio (MPR) = medication supply obtained/medication supply expected during follow-up period) were calculated. Results: Patients treated with Rebif® were younger, when compared with other Interferon Beta formulations (p < 0.01). The probability of switching to other DMTs was 60% higher for Betaferon®, 90% higher for Extavia®, and 110% higher for Plegridy®, when compared with Rebif® (p < 0.01). Plegridy® presented with 7% higher adherence (p < 0.01), and Betaferon® with 3% lower adherence (p = 0.03), when compared with Rebif®. The probability of MS-related hospital admissions was 40% higher in Avonex® (p = 0.03), 400% higher in Betaferon® (p < 0.01), and 60% higher in Plegridy® (p = 0.04), resulting into higher non-DMT-related costs, when compared with Rebif®. Discussion: Interferon Beta formulations presented with different prescription patterns, persistence, adherence, healthcare resource utilisation and costs, with Rebif® being used in younger patients and with less MS-related hospital admissions.