Aims: To evaluate the prevalence and time trends of gestational diabetes mellitus (GDM) across the five regions of Denmark with uniform national guidelines for screening and diagnosing GDM. Methods: This register-based national cohort study included 287,684 births from 2013 to 2017. Trends in GDM prevalence over time and differences between the five regions were evaluated. Crude and adjusted odd ratios (ORs) for GDM were calculated including potential confounding clinical risk factors as age, BMI, educational level, marital status, parity, country of origin and assisted reproduction. Results: From 2013 to 2017, GDM prevalence in Denmark increased by 7% per year (OR 1.07, 95% CI 1.06–1.09, P < 0.001). GDM prevalence varied considerably between regions and ranged from 3.0 to 5.9% in 2017, corresponding to a maximal regional difference of 97%. In crude analyses, the risk of GDM in 2017 was significantly different in four of five regions compared to the remaining regions (OR ranging from 0.60 to 1.55), and these differences persisted after adjusting for confounding clinical risk factors (adjusted OR: 0.59–1.45). Conclusion: The prevalence of GDM increased over time in all Danish regions with substantial regional divergence. Up to a 97%, difference in GDM prevalence was observed between Danish regions, which was not explained by available clinical risk factors. This occurred despite national guidelines and raises the question of whether regional variations in screening efficacy, diagnostic procedures or inequality in clinical health care access may explain the observed differences.