The feasibility of optical non-invasive evaluation of the graft function of vascular anastomosis was investigated in vitro using hyperspectral imaging (HSI) in the wavelength range from 500 to 600 nm. A Y-shaped vessel was made using porcine carotid arteries having an inner diameter of 3.5 to 4 mm. The graft vessel was anastomosed at a 45° angle with 8-0 polypropylene suture. Fresh porcine blood at an oxygen saturation of 100% was circulated in the specially designed circuit loop and through the graft or main vessel. The vessels were then irradiated with light, and the reflected light was captured with an HSI camera. The attenuation (A) image at each wavelength (λ) was obtained and the spectral A(λ) image was created. The spectral A(λ) image showed graft patency and changes in the hemoglobin concentration. The A(λ) decreased as the flow rate increased due to the orientation of the red blood cells. The experimental results indicated that imaging of the hemoglobin concentration without distortion from blood flow is possible using two wavelengths: 625 and 770 nm. This method is able to distinguish between the blood flow and changes in hemoglobin concentration. The multispectral and hyperspectral imaging method is useful for the non-invasive evaluation of graft function.