Introduction The diaphragm is the main muscle of inspiration, and its dysfunction contributes to adverse clinical outcomes in critically ill patients. We recently reported the infiltration of SARS-CoV-2, and the development of fibrosis, in the diaphragm of critically ill patients with COVID-19. In the current study, we aimed to characterise myofiber structure in the diaphragm of critically ill patients with COVID-19. Methods Diaphragm muscle specimens were collected during autopsy from patients who died of COVID-19 in three academic medical centres in the Netherlands in April and May 2020 (n=27). We studied diaphragm myofiber gene expression and structure and compared the findings obtained to those of deceased critically ill patients without COVID-19 (n=10). Results Myofibers of critically ill patients with COVID-19 showed on average larger cross-sectional area (slow-twitch myofibers: 2441±229 vs 1571±309 μm 2; fast-twitch myofibers: 1966±209 vs 1225±222 μm 2). Four critically ill patients with COVID-19 showed extremely large myofibers, which were splitting and contained many centralised nuclei. RNA-sequencing data revealed differentially expressed genes involved in muscle regeneration. Conclusion Diaphragm of critically ill patients with COVID-19 has distinct myopathic features compared with critically ill patients without COVID-19, which may contribute to the ongoing dyspnoea and fatigue in the patients surviving COVID-19 infection.