Abstract Background In 2015, a national research agenda was established for Dutch prehospital EMS to underpin the evidence base of care delivery and inform policymakers and funders. The continuously increasing demand for ambulance care and the reorientation towards the role of EMS in recent years may have changed research priorities. Therefore, this study aimed to update the Dutch national EMS research agenda. Methods A three-round online Delphi survey was used to explore and discuss different viewpoints and to reach consensus on research priorities (i.e., themes and special interest groups, e.g. patient types who require specific research attention). A multidisciplinary expert panel (n = 62) was recruited in the field of prehospital EMS and delegates of relevant professional organizations and stakeholders participated. In round one, fifty-nine research themes and six special interest groups (derived from several resources) were rated on importance on a 5-point scale by the panel members. In round two, the panel selected their priority themes and special interest groups (yes/no), and those with a positive difference score were further assessed in round three. In this final round, appropriateness of the remaining themes and agreement within the panel was taken into account, following the RAND/UCLA appropriateness method, which resulted in the final list of research priorities. Results The survey response per round varied between 94 and 100 percent. In round one, a reduction from 59 to 25 themes and the selection of three special interest groups was realized. Round two resulted in the prioritization of six themes and one special interest group ('Vulnerable elderly'). Round three showed an adequate level of agreement regarding all six themes: 'Registration and (digital) exchange of patient data in the chain of emergency care'; 'Mobile care consultation/Non conveyance'; 'Care coordination'; 'Cooperation with professional partners within the care domain'; 'Care differentiation' and 'Triage and urgency ...