This paper investigates the uncertainties underlying carbon footprint estimations of freight transportation, with a particular focus on trip-level shipment data. The research aimed to identify and classify possible causes of these uncertainties using Walker’s uncertainty matrix, drawing from literature reviews, protocol analyses, field research, and interviews. Findings suggest that the accuracy and transparency of assumptions are crucial when communicating carbon footprint estimations, as emphasized in current literature. This study contributes to the existing knowledge by providing deeper insights into the influence of uncertainty causes on the final estimation. The primary source of these uncertainties can largely be attributed to data accuracy in the calculation of carbon footprints. A structured approach is proposed to examine potential causes of uncertainties and their potential impacts on the final carbon footprint estimation. To demonstrate the effectiveness of this approach, a case study analyzing three freight movements to a building site was conducted. This research offers valuable insights and a systematic method for addressing uncertainties in carbon footprint estimations, ultimately supporting more informed decision-making in the freight transportation sector.