Limited reports have evaluated the utility of the popliteal vein (PV) specific to cannulation. The objective of this study was to characterize the diameter and length of the PV to evaluate this area as a potential cannulation site. The popliteal region in 23 formalin-embalmed, prosected donors was dissected, and the PV was exposed from the adductor hiatus (AH) superiorly to the small saphenous vein (SSV) inferiorly. The diameter of the popliteal vein was measured at the AH, SSV, and half of the distance from the AH to the SSV (MID) using a brass plumb bob. The length of the PV was measured to the AH, SSV, MID, and femoral condyles (FCs). Overall, the mean diameters and mean lengths for the combined population were calculated, as well as individual limbs (right, left) and anatomical sex. Univariate analysis used to evaluate differences in mean diameter and length measurements based on anatomical sex revealed significant differences (p < 0.05) for both diameter and length at all the landmarks evaluated. Multivariate analysis of PV diameter at the AH and SSV landmarks was statistically significant (p < 0.05) when laterally and anatomical comparing sex. These data provide full characterization of the PV in support of its utility in vascular access.