Objectives The primary objective of this investigation was to ultrasonographically evaluate the caudal vena cava to aorta (CVC:Ao) ratio in healthy, conscious cats and to generate reference intervals. A secondary objective was to identify the site of examination with the least intra- and inter-observer variability. This investigation was undertaken to assess whether the CVC:Ao ratio holds promise as a technique to assess intravascular volume responsiveness in cats. Methods In total, 42 healthy cats were included for reference interval generation. Ultrasound examinations were performed by two operators with each examination performed twice by each operator on the same occasion. Examinations were performed on conscious cats in left lateral recumbency. Ultrasound sites investigated were the subxiphoid, hepatic intercostal, hepatorenal and iliac bifurcation. Operators also assessed each site for ‘ease of visualisation’ on a scale of 0–3. Results Reference intervals were generated for the CVC:Ao ratio at all four ultrasonographic sites. While each site demonstrated low variability around its mean ratio, all sites exhibited significant intra- and inter-observer variability. The hepatorenal and iliac bifurcation sites were found to be the easiest to visualise (score 3; well-defined visualisation of both vessels) and had reference intervals of 0.8–1.41 and 0.75–1.2, respectively. Conclusions and relevance The ultrasonographic assessment of the CVC:Ao ratio was possible at four anatomical locations in the cat. The hepatorenal and iliac bifurcation may offer more readily assessable CVC:Ao ratios. Further studies are necessary to assess the utility of the CVC:Ao ratio in disease states, including in hypovolaemia and hypervolaemia.