Objectives The objectives of this study were to investigate recent trends in non‐invasive prenatal testing (NIPT) utilisation, including factors associated with geographical variation, and to determine whether maternal or regional characteristics are associated with uptake the of NIPT. Methods This retrospective cohort study included pregnant individuals in Ontario with an expected date of delivery from August 1st, 2016 to March 31st, 2020. Modified Poisson regression was used to estimate rate ratios for NIPT use adjusted for maternal and healthcare covariates. Results We found substantial variation in NIPT uptake between regions within the province. The highest uptake was found in urban areas, highest quintile of neighbourhood income and education, for those who were ≥40 years of age and had a history of previous aneuploidy, for those with a prenatal care visit in the first trimester, multiple pregnancy, multigravidity and body mass index within the normal range (18.5–24.9 kg/m2). Conclusion Our study demonstrated significant regional and maternal differences in NIPT uptake across Ontario. Given the large sample size and diverse population, our study may have implications for other jurisdictions with large, socio‐demographically and geographically diverse populations.